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Sample records for microdomains controlling carbohydrate-dependent

  1. Orientational control of block copolymer microdomains by sub-tesla magnetic fields

    NASA Astrophysics Data System (ADS)

    Gopinadhan, Manesh; Choo, Youngwoo; Feng, Xunda; Kawabata, Kohsuke; di, Xiaojun; Osuji, Chinedum

    Magnetic fields offer a versatile approach to controlling the orientation of block copolymer (BCP) microdomains during self-assembly. To date however, such control has required the imposition of large magnetic fields (>3T), necessitating the use of complex magnet systems - either superconducting or very large conventional resistive magnets. Here we demonstrate the ability to direct BCP self-assembly using considerably smaller fields (<1T) which are accessible using simple rare-earth permanent magnets. The low field alignment is enabled by the presence of small quantities of mesogenic species that are blended into, and co-assemble with the liquid crystalline (LC) mesophase of the side-chain LC BCP under study. In situ SAXS experiments reveal a pronounced dependence of the critical alignment field strength on the stoichiometry of the blend, and the ability to generate aligned microdomains with orientational distribution coefficients exceeding 0.95 at sub-1 T fields for appropriate stoichiometries. The alignment response overall can be rationalized in terms of increased mobility and grain size due to the presence of the mesogenic additive. We use a permanent magnet to fabricate films with aligned nanopores, and the utility of this approach to generate complex BCP microdomain patterns in thin films by local field screening are highlighted. NSF DMR-1410568 and DMR-0847534.

  2. Phase separation of lipid microdomains controlled by polymerized lipid bilayer matrices.

    PubMed

    Okazaki, Takashi; Tatsu, Yoshiro; Morigaki, Kenichi

    2010-03-16

    We developed a micropatterned model biological membrane on a solid substrate that can induce phase separation of lipid microdomains in a designed geometry. Micropatterned lipid bilayers were generated by the photolithographic polymerization of a diacetylene phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). By changing the UV dose for the photopolymerization, we could modulate the coverage of the surface by the polymeric bilayer domains. After removing nonpolymerized DiynePC, natural phospholipid membranes were incorporated into the micropatterned polymeric bilayer matrix by a self-assembly process (vesicle fusion). As we incorporated a ternary lipid mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM), and cholesterol (Chol) (1:1:1), liquid ordered domains (Lo: rich in SM and Chol) were accumulated in the polymer free regions, whereas liquid disordered domains (Ld: rich in DOPC) preferentially participated into the partially polymeric bilayer regions. It was postulated that Ld domains preferentially came in contact with the polymeric bilayer boundaries because of their lower elastic moduli and a smaller thickness mismatch at the boundary. The effect of polymeric bilayer matrix to hinder the size growth of Lo domains should also be playing an important role. The controlled phase separation should open new possibilities to locally concentrate membrane proteins and other nanometer-sized materials on the substrate by associating them with the lipid microdomains. PMID:20020734

  3. Controlling sub-microdomain structure in microphase-ordered block copolymers and their nanocomposites

    NASA Astrophysics Data System (ADS)

    Bowman, Michelle Kathleen

    Block copolymers exhibit a wealth of morphologies that continue to find ubiquitous use in a diverse variety of mature and emergent (nano)technologies, such as photonic crystals, integrated circuits, pharmaceutical encapsulents, fuel cells and separation membranes. While numerous studies have explored the effects of molecular confinement on such copolymers, relatively few have examined the sub-microdomain structure that develops upon modification of copolymer molecular architecture or physical incorporation of nanoscale objects. This work will address two relevant topics in this vein: (i) bidisperse brushes formed by single block copolymer molecules and (ii) copolymer nanocomposites formed by addition of molecular or nanoscale additives. In the first case, an isomorphic series of asymmetric poly(styrene-b -isoprene-b-styrene) (S1IS2) triblock copolymers of systematically varied chain length has been synthesized from a parent SI diblock copolymer. Small-angle x-ray scattering, coupled with dynamic rheology and self-consistent field theory (SCFT), reveals that the progressively grown S2 block initially resides in the I-rich matrix and effectively reduces the copolymer incompatibility until a critical length is reached. At this length, the S2 block co-locates with the S1 block so that the two blocks generate a bidisperse brush (insofar as the S1 and S2 lengths differ). This single-molecule analog to binary block copolymer blends affords unique opportunities for materials design at sub-microdomain length scales and provides insight into the transition from diblock to triblock copolymer (and thermoplastic elastomeric nature). In the second case, I explore the distribution of molecular and nanoscale additives in microphase-ordered block copolymers and demonstrate via SCFT that an interfacial excess, which depends strongly on additive concentration, selectivity and relative size, develops. These predictions are in agreement with experimental findings. Moreover, using a

  4. Control of Block Copolymer Microdomain Orientation from Solution using Electric Fields: Governing Parameters and Mechanisms

    NASA Astrophysics Data System (ADS)

    Böker, Alexander

    As nanotechnology increasingly gains importance in daily life, the need for novel nanoscopic structures also rises exponentially. For example, to keep up with Moore's law, the packing density of integrated circuits has to increase on an almost daily basis. Considering the growing number of electronically stored data, it is also clear that novel data storage techniques have to be devised aiming to increase the information density on a hard disk. For such applications, the microstructures formed by block copolymers via their microphase separation present an ideal template for the fabrication of nanoscale patterns ranging from 10 ? 100 nm [1]. In order to profit from the self-assembly of block copolymers into various microstructures, one has to be able to control the parameters that govern this unique self-ordering process. In addition, it would be desirable to guide selfassembly via external fields to form macroscopically oriented, highly ordered structures.

  5. Microdomains, Inflammation, and Atherosclerosis.

    PubMed

    Sorci-Thomas, Mary G; Thomas, Michael J

    2016-02-19

    Elevated levels of cholesteryl ester (CE)-enriched apoB containing plasma lipoproteins lead to increased foam cell formation, the first step in the development of atherosclerosis. Unregulated uptake of low-density lipoprotein cholesterol by circulating monocytes and other peripheral blood cells takes place through scavenger receptors and over time causes disruption in cellular cholesterol homeostasis. As lipoproteins are taken up, their CE core is hydrolyzed by liposomal lipases to generate free cholesterol (FC). FC can be either re-esterified and stored as CE droplets or shuttled to the plasma membrane for ATP-binding cassette transporter A1-mediated efflux. Because cholesterol is an essential component of all cellular membranes, some FC may be incorporated into microdomains or lipid rafts. These platforms are essential for receptor signaling and transduction, requiring rapid assembly and disassembly. ATP-binding cassette transporter A1 plays a major role in regulating microdomain cholesterol and is most efficient when lipid-poor apolipoprotein AI (apoAI) packages raft cholesterol into soluble particles that are eventually catabolized by the liver. If FC is not effluxed from the cell, it becomes esterified, CE droplets accumulate and microdomain cholesterol content becomes poorly regulated. This dysregulation leads to prolonged activation of immune cell signaling pathways, resulting in receptor oversensitization. The availability of apoAI or other amphipathic α-helix-rich apoproteins relieves the burden of excess microdomain cholesterol in immune cells allowing a reduction in immune cell proliferation and infiltration, thereby stimulating regression of foam cells in the artery. Therefore, cellular balance between FC and CE is essential for proper immune cell function and prevents chronic immune cell overstimulation and proliferation. PMID:26892966

  6. Functional microdomains in bacterial membranes.

    PubMed

    López, Daniel; Kolter, Roberto

    2010-09-01

    The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid--a known inhibitor of squalene synthases--impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated. PMID:20713508

  7. Functional microdomains in bacterial membranes

    PubMed Central

    López, Daniel; Kolter, Roberto

    2010-01-01

    The membranes of eukaryotic cells harbor microdomains known as lipid rafts that contain a variety of signaling and transport proteins. Here we show that bacterial membranes contain microdomains functionally similar to those of eukaryotic cells. These membrane microdomains from diverse bacteria harbor homologs of Flotillin-1, a eukaryotic protein found exclusively in lipid rafts, along with proteins involved in signaling and transport. Inhibition of lipid raft formation through the action of zaragozic acid—a known inhibitor of squalene synthases—impaired biofilm formation and protein secretion but not cell viability. The orchestration of physiological processes in microdomains may be a more widespread feature of membranes than previously appreciated. PMID:20713508

  8. Lipid Microdomains in Cell Nucleus

    PubMed Central

    Cascianelli, Giacomo; Villani, Maristella; Tosti, Marcello; Marini, Francesca; Bartoccini, Elisa; Viola Magni, Mariapia

    2008-01-01

    It is known that nuclear lipids play a role in proliferation, differentiation, and apoptotic process. Cellular nuclei contain high levels of phosphatidylcholine and sphingomyelin, which are partially linked with cholesterol and proteins to form lipid–protein complexes. These lipids are also associated with transcription factors and newly synthesized RNA but, up to date, their organization is still unknown. The aim of the present work was to study if these specific lipid–protein interactions could be nuclear membrane microdomains and to evaluate their possible role. The results obtained demonstrate for the first time the existence of nuclear microdomains characterized by a specific lipid composition similar to that of intranuclear lipid–protein complexes previously described. Nuclear microdomain lipid composition changes during cell proliferation when the content of newly synthesized RNA increases. Because previous data show a correlation between nuclear lipids and transcription process, the role of nuclear microdomains in cellular functions is discussed. PMID:18923143

  9. Stat-mediated Signaling Induced by Type I and Type II Interferons (IFNs) Is Differentially Controlled through Lipid Microdomain Association and Clathrin-dependent Endocytosis of IFN Receptors

    PubMed Central

    Marchetti, Marta; Monier, Marie-Noelle; Fradagrada, Alexandre; Mitchell, Keith; Baychelier, Florence; Eid, Pierre; Johannes, Ludger

    2006-01-01

    Type I (α/β) and type II (γ) interferons (IFNs) bind to distinct receptors, although they activate the same signal transducer and activator of transcription, Stat1, raising the question of how signal specificity is maintained. Here, we have characterized the sorting of IFN receptors (IFN-Rs) at the plasma membrane and the role it plays in IFN-dependent signaling and biological activities. We show that both IFN-α and IFN-γ receptors are internalized by a classical clathrin- and dynamin-dependent endocytic pathway. Although inhibition of clathrin-dependent endocytosis blocked the uptake of IFN-α and IFN-γ receptors, this inhibition only affected IFN-α–induced Stat1 and Stat2 signaling. Furthermore, the antiviral and antiproliferative activities induced by IFN-α but not IFN-γ were also affected. Finally, we show that, unlike IFN-α receptors, activated IFN-γ receptors rapidly become enriched in plasma membrane lipid microdomains. We conclude that IFN-R compartmentalization at the plasma membrane, through clathrin-dependent endocytosis and lipid-based microdomains, plays a critical role in the signaling and biological responses induced by IFNs and contributes to establishing specificity within the Jak/Stat signaling pathway. PMID:16624862

  10. Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium

    PubMed Central

    Schneider, Johannes; Klein, Teresa; Mielich-Süss, Benjamin; Koch, Gudrun; Franke, Christian; Kuipers, Oscar P.; Kovács, Ákos T.; Sauer, Markus; Lopez, Daniel

    2015-01-01

    Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. PMID:25909364

  11. Adenosine Receptors and Membrane Microdomains

    PubMed Central

    Lasley, Robert D.

    2010-01-01

    Adenosine receptors are a member of the large family of seven transmembrane spanning G protein coupled receptors (GPCR). The four adenosine receptor subtypes – A1, A2a, A2b, A3 – exert their effects via the activation of one or more heterotrimeric G proteins resulting in the modulation of intracellular signaling. Numerous studies over the past decade have documented the complexity of GPCR signaling at the level of protein-protein interactions as well as through signaling crosstalk. With respect to adenosine receptors the activation of one receptor subtype can have profound direct effects in one cell type, but little or no effect in other cells. There is significant evidence that the compartmentation of subcellular signaling plays a physiological role in the fidelity of GPCR signaling. This compartmentation is evident at the level of the plasma membrane in the form of membrane microdomains such as caveolae and lipid rafts. This review will summarize and critically assess our current understanding of the role of membrane microdomains in regulating adenosine receptor signaling. PMID:20888790

  12. Electron Propagation within Redox-Active Microdomains in Thin Films of Ferrocene-Containing Diblock Copolymers.

    PubMed

    Ghimire, Govinda; Yi, Yi; Derylo, Maksymilian A; Baker, Lane A; Ito, Takashi

    2015-11-10

    This paper reports the electrochemical behavior of redox-active microdomains in thin films of ferrocene-containing diblock copolymers, polystyrene-block-poly(2-(acryloyloxy)ethyl ferrocenecarboxylate) (PS-b-PAEFc). PS-b-PAEFc with different PAEFc volume fractions (PS154-b-PAEFc51, PS154-b-PAEFc26, and PS154-b-PAEFc12, where the subscripts represent the polymerization degree of each block; f(PAEFc) = 0.47, 0.30, and 0.17, respectively) was synthesized by sequential atom transfer radical polymerization. PS-b-PAEFc films of controlled thicknesses (20-160 nm) were prepared on gold substrates via spin-coating and characterized by ellipsometry. Microdomains were observed via atomic force microscopy on the surfaces of PS154-b-PAEFc51 and PS154-b-PAEFc26 thin films but not on the surfaces of PS154-b-PAEFc12 thin films. Electrochemical behavior of films was assessed by cyclic voltammetry and chronocoulometry in acetonitrile solution. The redox potential of ferrocene moieties was similar (ca. + 0.29 V vs Fc(+)/Fc) regardless of fPAEFc and film thickness. For PS154-b-PAEFc51 and PS154-b-PAEFc26, thicker films afforded larger faradaic peak currents and exhibited diffusion-controlled voltammograms at faster sweep rates. PS154-b-PAEFc26 produced voltammograms less influenced by solvent-induced swelling than PS154-b-PAEFc51, reflecting the improved morphological stability of PAEFc microdomains by redox-inert PS frameworks. In contrast, PS154-b-PAEFc12 films yielded similar faradaic peak currents regardless of film thickness and exhibited voltammograms indicative of surface-confined species. These observations suggest that PS154-b-PAEFc51 and PS154-b-PAEFc26 films contain continuous PAEFc microdomains extending from the electrode to the surface, in contrast to the PS154-b-PAEFc12 films which contain isolated PAEFc microdomains buried within the PS matrix. Electron propagation took place only through PAEFc microdomains that could electrically communicate with the underlying

  13. Microdomain Effects on Transverse Cardiac Propagation

    PubMed Central

    Lin, Joyce; Keener, James P.

    2014-01-01

    The effect of gap junctional coupling, sodium ion channel distribution, and extracellular conductivity on transverse conduction in cardiac tissue is explored using a microdomain model that incorporates aspects of the inhomogeneous cellular structure. The propagation velocities found in our model are compared to those in the classic bidomain model and indicate a strong ephaptic microdomain contribution to conduction depending on the parameter regime. We show that ephaptic effects can be quite significant in the junctional spaces between cells, and that the cell activation sequence is modified substantially by these effects. Further, we find that transverse propagation can be maintained by ephaptic effects, even in the absence of gap junctional coupling. The mechanism by which this occurs is found to be cablelike in that the junctional regions act like inverted cables. Our results provide insight into several recent experimental studies that indirectly indicate a mode of action potential propagation that does not rely exclusively on gap junctions. PMID:24559995

  14. Membrane microdomains: from seeing to understanding

    PubMed Central

    Truong-Quang, Binh-An; Lenne, Pierre-François

    2014-01-01

    The plasma membrane is a composite material, which forms a semi-permeable barrier and an interface for communication between the intracellular and extracellular environments. While the existence of membrane microdomains with nanoscale organization has been proved by the application of numerous biochemical and physical methods, direct observation of these heterogeneities using optical microscopy has remained challenging for decades, partly due to the optical diffraction limit, which restricts the resolution to ~200 nm. During the past years, new optical methods which circumvent this fundamental limit have emerged. Not only do these techniques allow direct visualization, but also quantitative characterization of nanoscopic structures. We discuss how these emerging optical methods have refined our knowledge of membrane microdomains and how they may shed light on the basic principles of the mesoscopic membrane organization. PMID:24600455

  15. Transmembrane voltage: Potential to induce lateral microdomains.

    PubMed

    Malinsky, Jan; Tanner, Widmar; Opekarova, Miroslava

    2016-08-01

    Lateral segregation of plasma membrane lipids is a generally accepted phenomenon. Lateral lipid microdomains of specific composition, structure and biological functions are established as a result of simultaneous action of several competing mechanisms which contribute to membrane organization. Various lines of evidence support the conclusion that among those mechanisms, the membrane potential plays significant and to some extent unique role. Above all, clear differences in the microdomain structure as revealed by fluorescence microscopy could be recognized between polarized and depolarized membranes. In addition, recent fluorescence spectroscopy experiments reported depolarization-induced changes in a membrane lipid order. In the context of earlier findings showing that plasma membranes of depolarized cells are less susceptible to detergents and the cells less sensitive to antibiotics or antimycotics treatment we discuss a model, in which membrane potential-driven re-organization of the microdomain structure contributes to maintaining membrane integrity during response to stress, pathogen attack and other challenges involving partial depolarization of the plasma membrane. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26902513

  16. Presence of detergent-resistant microdomains in lysosomal membranes.

    PubMed

    Taute, Antje; Wätzig, Kristin; Simons, Brigitte; Lohaus, Christiane; Meyer, Helmut; Hasilik, Andrej

    2002-10-18

    We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts. PMID:12379211

  17. A tunable and reversible platform for the intracellular formation of genetically engineered protein microdomains.

    PubMed

    Pastuszka, Martha K; Janib, Siti M; Weitzhandler, Isaac; Okamoto, Curtis T; Hamm-Alvarez, Sarah; Mackay, J Andrew

    2012-11-12

    From mitochondria to the nuclear envelope, the controlled assembly of micro- and nanostructures is essential for life; however, the level at which we can deliberately engineer the assembly of microstructures within intracellular environments remains primitive. To overcome this obstacle, we present a platform to reversibly assemble genetically engineered protein microdomains (GEPMs) on the time scale of minutes within living cells. Biologically inspired from the human protein tropoelastin, these protein polymers form a secondary aqueous phase above a tunable transition temperature. This assembly process is easily manipulated to occur at or near physiological temperature by adjusting molecular weight and hydrophobicity. We fused protein polymers to green fluorescent protein (GFP) to visualize their behavior within the cytoplasm. While soluble, these polymers have a similar intracellular diffusion constant as cytosolic proteins at 7.4 μm(2)/s; however, above their phase transition temperature, the proteins form distinct microdomains (0.1-2 μm) with a reduced diffusion coefficient of 1.1 μm(2)/s. Microdomain assembly and disassembly are both rapid processes with half-lives of 3.8 and 1.0 min, respectively. Via selection of the protein polymer, the assembly temperature is tunable between 20 and 40 °C. This approach may be useful to control intracellular formation of genetically engineered proteins and protein complexes into concentrated microdomains. PMID:23088632

  18. Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells

    PubMed Central

    Hatakeyama, Shingo; Sugihara, Kazuhiro; Nakayama, Jun; Akama, Tomoya O.; Wong, Shuk-Man Annie; Kawashima, Hiroto; Zhang, Jianing; Smith, David F.; Ohyama, Chikara; Fukuda, Minoru; Fukuda, Michiko N.

    2009-01-01

    Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1, Sfrs2, Sfrs5, and Sfrs7 gene products. Bacterially expressed Sfrs1 protein bound to B16-FTIII-M cells but not to parental B16 cells. Recombinant Sfrs1 protein bound to a series of fucosylated oligosaccharides in glycan array and plate-binding assays. When anti-Sfrs antibodies were injected intravenously into mice, antibodies labeled a subset of lung capillaries. Anti-Sfrs antibodies inhibited homing of I-peptide-displaying phage to the lung colonization of B16-FTIII-M cells in vivo in the mouse. These results strongly suggest that Sfrs proteins are responsible for fucosylated carbohydrate-dependent lung metastasis of epithelial cancers. PMID:19218444

  19. Carbohydrate-dependent binding of langerin to SodC, a cell wall glycoprotein of Mycobacterium leprae.

    PubMed

    Kim, Hee Jin; Brennan, Patrick J; Heaslip, Darragh; Udey, Mark C; Modlin, Robert L; Belisle, John T

    2015-02-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

  20. Carbohydrate-Dependent Binding of Langerin to SodC, a Cell Wall Glycoprotein of Mycobacterium leprae

    PubMed Central

    Kim, Hee Jin; Brennan, Patrick J.; Heaslip, Darragh; Udey, Mark C.; Modlin, Robert L.

    2014-01-01

    Langerhans cells participate in the immune response in leprosy by their ability to activate T cells that recognize the pathogen, Mycobacterium leprae, in a langerin-dependent manner. We hypothesized that langerin, the distinguishing C-type lectin of Langerhans cells, would recognize the highly mannosylated structures in pathogenic Mycobacterium spp. The coding region for the extracellular and neck domain of human langerin was cloned and expressed to produce a recombinant active trimeric form of human langerin (r-langerin). Binding assays performed in microtiter plates, by two-dimensional (2D) Western blotting, and by surface plasmon resonance demonstrated that r-langerin possessed carbohydrate-dependent affinity to glycoproteins in the cell wall of M. leprae. This lectin, however, yielded less binding to mannose-capped lipoarabinomannan (ManLAM) and even lower levels of binding to phosphatidylinositol mannosides. However, the superoxide dismutase C (SodC) protein of the M. leprae cell wall was identified as a langerin-reactive ligand. Tandem mass spectrometry verified the glycosylation of a recombinant form of M. leprae SodC (rSodC) produced in Mycobacterium smegmatis. Analysis of r-langerin affinity by surface plasmon resonance revealed a carbohydrate-dependent affinity of rSodC (equilibrium dissociation constant [KD] = 0.862 μM) that was 20-fold greater than for M. leprae ManLAM (KD = 18.69 μM). These data strongly suggest that a subset of the presumptively mannosylated M. leprae glycoproteins act as ligands for langerin and may facilitate the interaction of M. leprae with Langerhans cells. PMID:25422308

  1. Lipids and Membrane Microdomains in HIV-1 Replication

    PubMed Central

    Waheed, Abdul A.; Freed, Eric O.

    2009-01-01

    Several critical steps in the replication cycle of human immunodeficiency virus type 1 (HIV-1) – entry, assembly and budding – are complex processes that take place at the plasma membrane of the host cell. A growing body of data indicates that these early and late steps in HIV-1 replication take place in specialized plasma membrane microdomains, and that many of the viral and cellular components required for entry, assembly, and budding are concentrated in these microdomains. In particular, a number of studies have shown that cholesterol- and sphingolipid-enriched microdomains known as lipid rafts play important roles in multiple steps in the virus replication cycle. In this review, we provide an overview of what is currently known about the involvement of lipids and membrane microdomains in HIV-1 replication. PMID:19383519

  2. Lipid Microdomain Formation: Characterization by Infrared Spectroscopy and Ultrasonic Velocimetry

    PubMed Central

    Schultz, Zachary D.; Levin, Ira W.

    2008-01-01

    We demonstrate the use of vibrational infrared spectroscopy applied to characterize lipid microdomain sizes derived from a model raft-like system consisting of nonhydroxy galactocerebroside, cholesterol, and dipalmitoylphosphatidylcholine components. The resulting spectroscopic correlation field components of the lipid acyl chain CH2 methylene deformation modes, observed when lipid multilamellar assemblies are rapidly frozen from the liquid crystalline state to the gel phase, indicate the existence of lipid microdomains on a scale of several nanometers. The addition of cholesterol disrupts the glycosphingolipid selectively but perturbs the di-saturated chain phospholipid matrix. Complementary acoustic velocimetry measurements indicate that the microdomain formation decreases the total volume adiabatic compressibilities of the multilamellar vesicle assemblies. The addition of cholesterol, however, disrupts the galactocerebroside domains, resulting in a slight increase in the lipid assemblies' total adiabatic compressibility. The combination of these two physical approaches offers new insight into microdomain formation and their properties in model bilayer systems. PMID:18192352

  3. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

    PubMed Central

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-01-01

    Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  4. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

    PubMed

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-02-11

    Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  5. Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement

    PubMed Central

    Filipp, Dominik; Ballek, Ondrej; Manning, Jasper

    2012-01-01

    In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering. PMID:22701458

  6. Graphoepitaxy of diblock-copolymers microdomains with chemical patterns

    NASA Astrophysics Data System (ADS)

    Checco, Antonio; Ocko, Benjamin M.; Misner, Matthew; Xu, Ji; Russell, Thomas P.

    2007-03-01

    Topographically patterned substrates have been used in recent years to laterally confine diblock copolymer (DBC) thin films in order to induce long-range lateral order of the DBC microdomain lattice with respect to a macroscopic reference. Here we demonstrate that surfaces with pure chemical patterns can be used to confine laterally diblock copolymers thin films through template-induced dewetting. A thin DBC film (PS-PEO) is spun cast on top of a surface chemically patterned with micron-sized, wettable domains prepared by oxidative nanolithography. Subsequently, annealing is used to direct the dewetting of the thin film into regions which are conformal to the patterns. We investigate the conditions (film thickness, annealing time) necessary to obtain dewetted structures reproducing the pattern shape with a high level of fidelity. In addition, we study the effect of pattern shape and size on the long-range order of DBC microdomains.

  7. Determination of microdomain size of hydrophobic polyelectrolytes by luminescence quenching

    SciTech Connect

    Strauss, U.P.; Zhong, Y.; Zdanowicz, V.S.

    1993-12-31

    The size of the hydrophobic microdomains of a hydrolyzed copolymer of maleic anhydride and hexyl vinyl ether has been measured in aqueous lithium chloride solutions by luminescence quenching using a photon counting technique. Several probe-quencher combinations were employed, including tris(2,2`-bipyridine)ruthenium(II) with 9-methylanthracene, pyrene with benzophenone, and pyrene with nonyl-phenyl ketone. For the last of these, the number of repeat units per microdomain was found to be 46, irrespective of polyacid concentration or extent of micellization due to variations in pH. With the other probe-quencher systems approximately the same number was obtained at pH 4.5 where the polyacid is close to completely micellized. At higher pH values, where micellization is incomplete, special effects were observed which are ascribed to nonmicellar binding of probe or quencher.

  8. Membrane-Sculpting BAR Domains Generate Stable Lipid Microdomains

    PubMed Central

    Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V.; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G.; Lappalainen, Pekka

    2014-01-01

    SUMMARY Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by “freezing” phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. PMID:24055060

  9. Glutamate Receptor Dynamics in Dendritic Microdomains

    PubMed Central

    Newpher, Thomas M.; Ehlers, Michael D.

    2008-01-01

    Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity. PMID:18498731

  10. A microdomain for protein secretion in Gram-positive bacteria.

    PubMed

    Rosch, Jason; Caparon, Michael

    2004-06-01

    Gram-positive bacteria face unique challenges in generating biologically active conformations for their exported proteins because they lack a dedicated compartment for folding secreted polypeptides. We have discovered that protein secretion by way of the general secretory (Sec) pathway in the important human pathogen Streptococcus pyogenes proceeds through a single microdomain. Unlike other mechanisms for asymmetry involving the Sec pathway, proteins destined for secretion are targeted to a single locus distal to either cell pole that has specialized to contain the Sec translocons. This subcellular organization may represent a paradigm for secretion common to other Gram-positive pathogens with profound implications for pathogenesis. PMID:15178803

  11. Analysis of lipid-composition changes in plasma membrane microdomains[S

    PubMed Central

    Ogiso, Hideo; Taniguchi, Makoto; Okazaki, Toshiro

    2015-01-01

    Sphingolipids accumulate in plasma membrane microdomain sites, such as caveolae or lipid rafts. Such microdomains are considered to be important nexuses for signal transduction, although changes in the microdomain lipid components brought about by signaling are poorly understood. Here, we applied a cationic colloidal silica bead method to analyze plasma membrane lipids from monolayer cells cultured in a 10 cm dish. The detergent-resistant fraction from the silica bead-coated membrane was analyzed by LC-MS/MS to evaluate the microdomain lipids. This method revealed that glycosphingolipids composed the microdomains as a substitute for sphingomyelin (SM) in mouse embryonic fibroblasts (tMEFs) from an SM synthase 1/2 double KO (DKO) mouse. The rate of formation of the detergent-resistant region was unchanged compared with that of WT-tMEFs. C2-ceramide (Cer) stimulation caused greater elevations in diacylglycerol and phosphatidic acid levels than in Cer levels within the microdomains of WT-tMEFs. We also found that lipid changes in the microdomains of SM-deficient DKO-tMEFs caused by serum stimulation occurred in the same manner as that of WT-tMEFs. This practical method for analyzing membrane lipids will facilitate future comprehensive analyses of membrane microdomain-associated responses. PMID:26116739

  12. Small dedifferentiated cardiomyocytes bordering on microdomains of fibrosis: evidence for reverse remodeling with assisted recovery.

    PubMed

    Al Darazi, Fahed; Zhao, Wenyuan; Zhao, Tieqiang; Sun, Yao; Marion, Tony N; Ahokas, Robert A; Bhattacharya, Syamal K; Gerling, Ivan C; Weber, Karl T

    2014-09-01

    With the perspective of functional myocardial regeneration, we investigated small cardiomyocytes bordering on microdomains of fibrosis, where they are dedifferentiated re-expressing fetal genes, and determined: (1) whether they are atrophied segments of the myofiber syncytium, (2) their redox state, (3) their anatomic relationship to activated myofibroblasts (myoFb), given their putative regulatory role in myocyte dedifferentiation and redifferentiation, (4) the relevance of proteolytic ligases of the ubiquitin-proteasome system as a mechanistic link to their size, and (5) whether they could be rescued from their dedifferentiated phenotype. Chronic aldosterone/salt treatment (ALDOST) was invoked, where hypertensive heart disease with attendant myocardial fibrosis creates the fibrillar collagen substrate for myocyte sequestration, with propensity for disuse atrophy, activated myoFb, and oxidative stress. To address phenotype rescue, 4 weeks of ALDOST was terminated followed by 4 weeks of neurohormonal withdrawal combined with a regimen of exogenous antioxidants, ZnSO4, and nebivolol (assisted recovery). Compared with controls, at 4 weeks of ALDOST, we found small myocytes to be: (1) sequestered by collagen fibrils emanating from microdomains of fibrosis and representing atrophic segments of the myofiber syncytia, (2) dedifferentiated re-expressing fetal genes (β-myosin heavy chain and atrial natriuretic peptide), (3) proximal to activated myoFb expressing α-smooth muscle actin microfilaments and angiotensin-converting enzyme, (4) expressing reactive oxygen species and nitric oxide with increased tissue 8-isoprostane, coupled to ventricular diastolic and systolic dysfunction, and (5) associated with upregulated redox-sensitive proteolytic ligases MuRF1 and atrogin-1. In a separate study, we did not find evidence of myocyte replication (BrdU labeling) or expression of stem cell antigen (c-Kit) at weeks 1-4 ALDOST. Assisted recovery caused complete disappearance of

  13. SMALL DEDIFFERENTIATED CARDIOMYOCYTES BORDERING ON MICRODOMAINS OF FIBROSIS: EVIDENCE FOR REVERSE REMODELING WITH ASSISTED RECOVERY

    PubMed Central

    Al Darazi, Fahed; Zhao, Wenyuan; Zhao, Tieqiang; Sun, Yao; Marion, Tony N.; Ahokas, Robert A.; Bhattacharya, Syamal K.; Gerling, Ivan C.; Weber, Karl T.

    2014-01-01

    With the perspective of functional myocardial regeneration, we investigated small cardiomyocytes bordering on microdomains of fibrosis, where they are dedifferentiated re-expressing fetal genes, and determined: i) whether they are atrophied segments of the myofiber syncytium; ii) their redox state; iii) their anatomic relationship to activated myofibroblasts (myoFb), given their putative regulatory role in myocyte dedifferentiation and re-differentiation; iv) the relevance of proteolytic ligases of the ubiquitin-proteasome system (UPS) as a mechanistic link to their size; and v) whether they could be rescued from their dedifferentiated phenotype. Chronic aldosterone/salt treatment (ALDOST) was invoked, where hypertensive heart disease with attendant myocardial fibrosis creates the fibrillar collagen substrate for myocyte sequestration with propensity for disuse atrophy, activated myoFb and oxidative stress. To address phenotype rescue, 4 wks ALDOST was terminated followed by 4 wks neurohormonal withdrawal combined with a regimen of exogenous antioxidants, ZnSO4 and nebivolol (assisted recovery). Compared to controls, at 4 wks ALDOST we found small myocytes to be: 1) sequestered by collagen fibrils emanating from microdomains of fibrosis and representing atrophic segments of the myofiber syncytia; 2) dedifferentiated re-expressing fetal genes (β-myosin heavy chain and atrial natriuretic peptide); 3) proximal to activated myoFb expressing α-smooth muscle actin microfilaments and angiotensin-converting enzyme; 4) expressing reactive oxygen species and nitric oxide (NO) with increased tissue 8-isoprostane, coupled to ventricular diastolic and systolic dysfunction; and 5) associated with upregulated redox-sensitive, proteolytic ligases MuRF1 and atrogin-1. In a separate study, we did not find evidence of myocyte replication (BrdU labeling) or expression of stem cell antigen (c-Kit) at wks 1-4 ALDOST. Assisted recovery caused: complete disappearance of myoFb from sites

  14. Orientations of Diblock Copolymer Microdomains at Different Film Thicknesses

    NASA Astrophysics Data System (ADS)

    Chaikin, Paul; Park, Miri; Harrison, Christopher; Register, Richard; Adamson, Doug

    1996-03-01

    We prepared films with a range of thicknesses (50-300 nm) of a styrene-butadiene diblock copolymer, synthesized to produce a cylindrical morphology. Solutions of different polymer concentrations in toluene were spun onto carbon-coated glass slides. The films were then placed onto a Transmission Electron Microscope (TEM) grid by water lift-off, annealed, stained with osmium tetraoxide, and examined with a TEM. Over a wide range of film thicknesses, the cylinders lie parallel to the substrate. We present preliminary results that show a cylinder orientation perpendicular to the substrate at a thickness of many microdomain spacings. We speculate that the alignment mechanism is different from that found in a previous study of Kraton D1102(M. A. van Dijk and R. van den Berg, Macromolecules 28), 6773 (1995) which shows a perpendicular orientation with spin-coated films, but for a film thickness between one and two microdomain spacings. This work was supported by the NSF under DMR 9400362.

  15. Correlative infrared nanospectroscopic and nanomechanical imaging of block copolymer microdomains.

    PubMed

    Pollard, Benjamin; Raschke, Markus B

    2016-01-01

    Intermolecular interactions and nanoscale phase separation govern the properties of many molecular soft-matter systems. Here, we combine infrared vibrational scattering scanning near-field optical microscopy (IR s-SNOM) with force-distance spectroscopy for simultaneous characterization of both nanoscale optical and nanomechanical molecular properties through hybrid imaging. The resulting multichannel images and correlative analysis of chemical composition, spectral IR line shape, modulus, adhesion, deformation, and dissipation acquired for a thin film of a nanophase separated block copolymer (PS-b-PMMA) reveal complex structural variations, in particular at domain interfaces, not resolved in any individual signal channel alone. These variations suggest that regions of multicomponent chemical composition, such as the interfacial mixing regions between microdomains, are correlated with high spatial heterogeneity in nanoscale material properties. PMID:27335750

  16. Correlative infrared nanospectroscopic and nanomechanical imaging of block copolymer microdomains

    PubMed Central

    Pollard, Benjamin

    2016-01-01

    Summary Intermolecular interactions and nanoscale phase separation govern the properties of many molecular soft-matter systems. Here, we combine infrared vibrational scattering scanning near-field optical microscopy (IR s-SNOM) with force–distance spectroscopy for simultaneous characterization of both nanoscale optical and nanomechanical molecular properties through hybrid imaging. The resulting multichannel images and correlative analysis of chemical composition, spectral IR line shape, modulus, adhesion, deformation, and dissipation acquired for a thin film of a nanophase separated block copolymer (PS-b-PMMA) reveal complex structural variations, in particular at domain interfaces, not resolved in any individual signal channel alone. These variations suggest that regions of multicomponent chemical composition, such as the interfacial mixing regions between microdomains, are correlated with high spatial heterogeneity in nanoscale material properties. PMID:27335750

  17. Kinetics of Diffusing Polymer Encounter in Confined Cellular Microdomains

    NASA Astrophysics Data System (ADS)

    Amitai, A.; Kupka, I.; Holcman, D.

    2013-12-01

    We study the mean first time that two monomers, located on the same polymer, encounter in a confined microdomain. Approximating the confined geometry by a harmonic potential well, we obtain an asymptotic expression for the mean first encounter time (MFETC) as a function of the radius ɛ around one monomer. By studying the end-to-end distance of the polymer in a ball using the Edwards' formalism, we derive an other estimation of the MFETC. We validate the asymptotic formulas using Brownian simulations and derive their range of validity in terms of the polymer length. We apply the present models to compute the mean time for a gene located far away from a promoter site to be activated during looping in confined genomic territories.

  18. Molecular organization of cholesterol in polyunsaturated membranes: microdomain formation.

    PubMed Central

    Brzustowicz, Michael R; Cherezov, Vadim; Caffrey, Martin; Stillwell, William; Wassall, Stephen R

    2002-01-01

    The molecular organization of cholesterol in phospholipid bilayers composed of 1,2-diarachidonylphosphatidylcholine (20:4-20:4PC), 1-stearoyl-2-arachidonylphosphatidylcholine (18:0-20:4PC), and 20:4-20:4PC/18:0-20:4PC (1/1 mol) was investigated by solid-state (2)H NMR and by low- and wide-angle x-ray diffraction (XRD). On the basis of distinct quadrupolar powder patterns arising from [3 alpha-(2)H(1)]cholesterol intercalated into the membrane and phase separated as solid, solubility chi(NMR)(chol) = 17 +/- 2 mol% and tilt angle alpha(0) = 25 +/- 1 degrees in 20:4-20:4PC were determined. The corresponding values in 18:0-20:4PC were chi (NMR)(chol) > or = 50 mol% and alpha(0) = 16 +/- 1 degrees. Cholesterol solubility determined by XRD was chi(NMR)(chol) = 15 +/- 2 mol% and chi(NMR)(chol) = 49 +/- 1 mol% for 20:4-20:4PC and 18:0-20:4PC, respectively. XRD experiments show that the solid sterol is monohydrate crystals presumably residing outside the bilayer. The (2)H NMR spectrum for equimolar [3 alpha-(2)H(1)]cholesterol added to mixed 20:4-20:4PC/18:0-20:4PC (1/1 mol) membranes is consistent with segregation of cholesterol into 20:4-20:4PC and 18:0-20:4PC microdomains of <160 A in size that preserve the molecular organization of sterol in the individual phospholipid constituents. Our results demonstrate unambiguously that cholesterol has low affinity to polyunsaturated fatty acids and support hypotheses of lateral phase separation of membrane constituents into sterol-poor/polyunsaturated fatty acid-rich and sterol-rich/saturated fatty acid-rich microdomains. PMID:11751316

  19. Contribution of complement component C3 and complement receptor type 3 to carbohydrate-dependent uptake of oligomannose-coated liposomes by peritoneal macrophages.

    PubMed

    Abe, Yu; Kuroda, Yasuhiro; Kuboki, Noritaka; Matsushita, Misao; Yokoyama, Naoaki; Kojima, Naoya

    2008-11-01

    Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3. PMID:18694897

  20. Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

    SciTech Connect

    Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko . E-mail: iwashita@tmig.or.jp

    2006-12-22

    The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with {beta}-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane.

  1. Increased Gsα within Blood Cell Membrane Lipid Microdomains in Some Depressive Disorders: An Exploratory Study

    PubMed Central

    Mooney, John J.; Samson, Jacqueline A.; McHale, Nancy L.; Pappalarado, Kathleen M.; Alpert, Jonathan E.; Schildkraut, Joseph J.

    2013-01-01

    The stimulatory guanine nucleotide binding protein Gs couples many cellular receptors to adenylate cyclase, and the Gsα subunit activates all 9 isoforms of the adenylate cyclase catalytic unit to produce the enzyme product cyclicAMP or cAMP. In prefrontal cortex and cerebellum of unipolar depressive suicides, Rasenick and colleagues have found increased concentrations of Gsα in membrane lipid microdomains (Donati et al, 2008), where the ensconced Gsα is less likely to activate adenylate cyclase by receptor and postreceptor pathways (Allen et al, 2005 & 2009). We report that a group of 7 depressed patients (DP-1) had (1) reduced activation of platelet receptor-stimulated adenylate cyclase by both prostaglandins E2 and D2 compared to controls, and (2) reduced postreceptor stimulation of adenylate cyclase by aluminum fluoride ion in both platelets and mononuclear leukocytes when compared to both another group of depressed patients (DP-2, n=17) and to controls (n=21). Our observations in the blood cells of the group DP-1 support the findings of Donati et al (2008), and they reflect the importance of this interaction between the activated Gsα subunit and membrane lipid microdomains in the pathophysiology and treatment of some major depressive disorders. PMID:23490066

  2. The glycosylation pattern of common allergens: the recognition and uptake of Der p 1 by epithelial and dendritic cells is carbohydrate dependent.

    PubMed

    Al-Ghouleh, Abeer; Johal, Ramneek; Sharquie, Inas K; Emara, Mohammed; Harrington, Helen; Shakib, Farouk; Ghaemmaghami, Amir M

    2012-01-01

    Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1-2, 1-3 and 1-6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production. PMID:22479478

  3. The Glycosylation Pattern of Common Allergens: The Recognition and Uptake of Der p 1 by Epithelial and Dendritic Cells Is Carbohydrate Dependent

    PubMed Central

    Al-Ghouleh, Abeer; Johal, Ramneek; Sharquie, Inas K.; Emara, Mohammed; Harrington, Helen; Shakib, Farouk; Ghaemmaghami, Amir M.

    2012-01-01

    Allergens are initiators of both innate and adaptive immune responses. They are recognised at the site of entry by epithelial and dendritic cells (DCs), both of which activate innate inflammatory circuits that can collectively induce Th2 immune responses. In an attempt to have a better understanding of the role of carbohydrates in the recognition and uptake of allergens by the innate immune system, we defined common glycosylation patterns in major allergens. This was done using labelled lectins and showed that allergens like Der p 1 (Dermatophagoides pteronyssinus group 1), Fel d 1 (Felis domisticus), Ara h 1 (Arachis hypogaea), Der p 2 (Dermatophagoides pteronyssinus group 2), Bla g 2 (Blattella germanica) and Can f 1 (Canis familiaris) are glycosylated and that the main dominant sugars on these allergens are 1–2, 1–3 and 1–6 mannose. These observations are in line with recent reports implicating the mannose receptor (MR) in allergen recognition and uptake by DCs and suggesting a major link between glycosylation and allergen recognition. We then looked at TSLP (Thymic Stromal Lymphopoietin) cytokine secretion by lung epithelia upon encountering natural Der p 1 allergen. TSLP is suggested to drive DC maturation in support of allergic hypersensitivity reactions. Our data showed an increase in TSLP secretion by lung epithelia upon stimulation with natural Der p 1 which was carbohydrate dependent. The deglycosylated preparation of Der p 1 exhibited minimal uptake by DCs compared to the natural and hyperglycosylated recombinant counterparts, with the latter being taken up more readily than the other preparations. Collectively, our data indicate that carbohydrate moieties on allergens play a vital role in their recognition by innate immune cells, implicating them in downstream deleterious Th2 cell activation and IgE production. PMID:22479478

  4. Molecular microdomains in a sensory terminal, the vestibular calyx ending.

    PubMed

    Lysakowski, Anna; Gaboyard-Niay, Sophie; Calin-Jageman, Irina; Chatlani, Shilpa; Price, Steven D; Eatock, Ruth Anne

    2011-07-01

    Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent's central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology. PMID:21734302

  5. P2Y₁ receptor-dependent diacylglycerol signaling microdomains in β cells promote insulin secretion.

    PubMed

    Wuttke, Anne; Idevall-Hagren, Olof; Tengholm, Anders

    2013-04-01

    Diacylglycerol (DAG) controls numerous cell functions by regulating the localization of C1-domain-containing proteins, including protein kinase C (PKC), but little is known about the spatiotemporal dynamics of the lipid. Here, we explored plasma membrane DAG dynamics in pancreatic β cells and determined whether DAG signaling is involved in secretagogue-induced pulsatile release of insulin. Single MIN6 cells, primary mouse β cells, and human β cells within intact islets were transfected with translocation biosensors for DAG, PKC activity, or insulin secretion and imaged with total internal reflection fluorescence microscopy. Muscarinic receptor stimulation triggered stable, homogenous DAG elevations, whereas glucose induced short-lived (7.1 ± 0.4 s) but high-amplitude elevations (up to 109 ± 10% fluorescence increase) in spatially confined membrane regions. The spiking was mimicked by membrane depolarization and suppressed after inhibition of exocytosis or of purinergic P2Y₁, but not P2X receptors, reflecting involvement of autocrine purinoceptor activation after exocytotic release of ATP. Each DAG spike caused local PKC activation with resulting dissociation of its substrate protein MARCKS from the plasma membrane. Inhibition of spiking reduced glucose-induced pulsatile insulin secretion. Thus, stimulus-specific DAG signaling patterns appear in the plasma membrane, including distinct microdomains, which have implications for the kinetic control of exocytosis and other membrane-associated processes. PMID:23299857

  6. Regulation of cellular communication by signaling microdomains in the blood vessel wall.

    PubMed

    Billaud, Marie; Lohman, Alexander W; Johnstone, Scott R; Biwer, Lauren A; Mutchler, Stephanie; Isakson, Brant E

    2014-01-01

    It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

  7. Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

    PubMed Central

    Billaud, Marie; Lohman, Alexander W.; Johnstone, Scott R.; Biwer, Lauren A.; Mutchler, Stephanie; Isakson, Brant E.

    2014-01-01

    It has become increasingly clear that the accumulation of proteins in specific regions of the plasma membrane can facilitate cellular communication. These regions, termed signaling microdomains, are found throughout the blood vessel wall where cellular communication, both within and between cell types, must be tightly regulated to maintain proper vascular function. We will define a cellular signaling microdomain and apply this definition to the plethora of means by which cellular communication has been hypothesized to occur in the blood vessel wall. To that end, we make a case for three broad areas of cellular communication where signaling microdomains could play an important role: 1) paracrine release of free radicals and gaseous molecules such as nitric oxide and reactive oxygen species; 2) role of ion channels including gap junctions and potassium channels, especially those associated with the endothelium-derived hyperpolarization mediated signaling, and lastly, 3) mechanism of exocytosis that has considerable oversight by signaling microdomains, especially those associated with the release of von Willebrand factor. When summed, we believe that it is clear that the organization and regulation of signaling microdomains is an essential component to vessel wall function. PMID:24671377

  8. The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

    PubMed Central

    Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

    2008-01-01

    Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

  9. FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains

    PubMed Central

    Zamaleeva, Alsu I.; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne

    2015-01-01

    Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging. PMID:26404317

  10. Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling.

    PubMed

    Kock, Christian; Arlt, Henning; Ungermann, Christian; Heinisch, Jürgen J

    2016-09-01

    The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate 'membrane compartment occupied by Wsc1' (MCW) shows moderate overlap with other Wsc-type sensors, but not with those of the Mid-type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine-rich head group near the N-terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis. PMID:27337501

  11. Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms.

    PubMed

    Aldeek, Fadi; Mustin, Christian; Balan, Lavinia; Roques-Carmes, Thibault; Fontaine-Aupart, Marie-Pierre; Schneider, Raphaël

    2011-08-01

    Quantum dots (QDs) nanoprobes are emerging as alternatives to small-molecule fluorescent probes in biomedical technology. This paper reports an efficient and rapid method of producing highly dispersed and stable CdSe-core QDs with a hydrophobic gradient. Amphiphilic core/shell CdSe/ZnS QDs were prepared by ligand exchange at the surface of lipophilic CdSe/ZnS QDs using the dihydrolipoic acid (DHLA) dithiol ligand linked to leucine or phenylalanine amino acids. Contact angle relaxations on a hydrophobic surface and surface tension measurements indicated that aqueous dispersions of CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs exhibit increased hydrophobicity compared to CdSe-core QDs capped by the hydrophilic 3-mercaptopropionic acid (MPA) ligand. We found that the surface functional groups and the ligand density at the periphery of these QDs significantly dictated their interactions with a complex biological matrix called biofilm. Using fluorescence confocal microscopy and an autocorrelation function (semi-variogram), we demonstrated that MPA-capped QDs were homogeneously associated to the biopolymers, while amphiphilic CdSe/ZnS@DHLA-Leu or CdSe/ZnS@DHLA-Phe QDs were specifically confined allowing identification of hydrophobic microdomains of the biofilms. Results obtained clearly point out that the final destination of QDs in biofilms can properly be controlled by an appropriate design of surface ligands. PMID:21549423

  12. Influence of Homopolymers on the Microdomain Behavior of Block Copolymers in 2D Confinement

    NASA Astrophysics Data System (ADS)

    Kim, Youngkeol; Hwang, Sungyoul; Yu, Guiduk; Char, Kookheon

    Constraints imposed by nanometer scale confinement lead to changes in bulk equilibrium behavior of block copolymers (BCPs). Cylindrical pores with diameters corresponding to the length equivalent of several copolymer chains have been employed to investigate the influence of two-dimensional confinement on the behavior of BCPs. In this study, we expand the scope to homopolymer-BCP binary blends. Given fraction of homopolymers, the phase behavior of blends is dependent on molecular weight (Mw) of homopolymers. Lamella- and cylinder-forming poly(styrene-b-butadiene) (PS-b-PBD) and PS homopolymers (hPS) were drawn into the pores of anodized aluminum oxide (AAO) membranes in the melt by capillary forces. Based on the detailed observation of the morphologies within porous columns, we analyzed the structural transition of BCPs induced by the presence of hPS and confinement. The effect of hPS on the micro-domain of BCPs is greatly accentuated in nanoscale confinement compared to the bulk state due to the entropic loss of polymer chains. Pore diameters of AAO and Mw of the PS-b-PBD are also controlled so as to examine the effects of confinement on the phase transition of PS-b-PBD/hPS blends.

  13. Formation and preservation of biotite-rich microdomains in high-temperature rocks from the Antananarivo Block, Madagascar

    NASA Astrophysics Data System (ADS)

    Cenki-Tok, Bénédicte; Berger, Alfons; Gueydan, Frédéric

    2016-07-01

    Highly restitic rocks from the Antananarivo Block in northern Madagascar are investigated in this study in order to unravel processes of H2O-rich biotite formation in HT rocks. Polyphase metamorphism and melt migration occurred at 0.6 GPa and 850 °C. Biotite remains stable together with orthopyroxene and makes up to 45 vol% of the rock. In addition, three well-characterised and delimited microdomains having different textural, chemical and petrological characteristics are preserved. Thermodynamic models using the specific bulk compositions of the domains are in agreement with petrological observations. These rocks provide evidence that the lower crust may be strongly heterogeneous, locally associated to the formation of hydrous restites controlled by episodes of melt production and melt escape. This has significant consequences for understanding of the lower crust.

  14. cAMP signaling microdomains and their observation by optical methods

    PubMed Central

    Calebiro, Davide; Maiellaro, Isabella

    2014-01-01

    The second messenger cyclic AMP (cAMP) is a major intracellular mediator of many hormones and neurotransmitters and regulates a myriad of cell functions, including synaptic plasticity in neurons. Whereas cAMP can freely diffuse in the cytosol, a growing body of evidence suggests the formation of cAMP gradients and microdomains near the sites of cAMP production, where cAMP signals remain apparently confined. The mechanisms responsible for the formation of such microdomains are subject of intensive investigation. The development of optical methods based on fluorescence resonance energy transfer (FRET), which allow a direct observation of cAMP signaling with high temporal and spatial resolution, is playing a fundamental role in elucidating the nature of such microdomains. Here, we will review the optical methods used for monitoring cAMP and protein kinase A (PKA) signaling in living cells, providing some examples of their application in neurons, and will discuss the major hypotheses on the formation of cAMP/PKA microdomains. PMID:25389388

  15. Flipping the Switch on Clathrin-Mediated Endocytosis using Thermally Responsive Protein Microdomains

    PubMed Central

    Pastuszka, Martha K.; Okamoto, Curtis T.; Hamm-Alvarez, Sarah F.

    2014-01-01

    A ubiquitous approach to study protein function is to knock down activity (gene deletions, siRNA, small molecule inhibitors, etc) and study the cellular effects. Using a new methodology, this manuscript describes how to rapidly and specifically switch off cellular pathways using thermally responsive protein polymers. A small increase in temperature stimulates cytosolic elastin-like polypeptides (ELPs) to assemble microdomains. We hypothesize that ELPs fused to a key effector in a target macromolecular complex will sequester the complex within these microdomains, which will bring the pathway to a halt. To test this hypothesis, we fused ELPs to clathrin-light chain (CLC), a protein associated with clathrin-mediated endocytosis. Prior to thermal stimulation, the ELP fusion is soluble and clathrin-mediated endocytosis remains ‘on.’ Increasing the temperature induces the assembly of ELP fusion proteins into organelle-sized microdomains that switches clathrin-mediated endocytosis ‘off.’ These microdomains can be thermally activated and inactivated within minutes, are reversible, do not require exogenous chemical stimulation, and are specific for components trafficked within the clathrin-mediated endocytosis pathway. This temperature-triggered cell switch system represents a new platform for the temporal manipulation of trafficking mechanisms in normal and disease cell models and has applications for manipulating other intracellular pathways. PMID:25419208

  16. Hypertrophy, gene expression, and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D

    PubMed Central

    Curtis, Matthew W.; Sharma, Sadhana; Desai, Tejal A.

    2011-01-01

    Cardiac myocytes are known to be influenced by the rigidity and topography of their physical microenvironment. It was hypothesized that 3D heterogeneity introduced by purely physical microdomains regulates cardiac myocyte size and contraction. This was tested in vitro using polymeric microstructures (G′=1.66 GPa) suspended with random orientation in 3D by a soft Matrigel matrix (G′=22.9 Pa). After 10 days of culture, the presence of 100 μm-long microstructures in 3D gels induced fold increases in neonatal rat ventricular myocyte size (1.61±0.06, p<0.01) and total protein/cell ratios (1.43± 0.08, p<0.05) that were comparable to those induced chemically by 50 μM phenylephrine treatment. Upon attachment to microstructures, individual myocytes also had larger cross-sectional areas (1.57±0.05, p<0.01) and higher average rates of spontaneous contraction (2.01±0.08, p<0.01) than unattached myocytes. Furthermore, the inclusion of microstructures in myocyte-seeded gels caused significant increases in the expression of beta-1 adrenergic receptor (β1-AR, 1.19±0.01), cardiac ankyrin repeat protein (CARP, 1.26±0.02), and sarcoplasmic reticulum calcium-ATPase (SERCA2, 1.59±0.12, p<0.05), genes implicated in hypertrophy and contractile activity. Together, the results demonstrate that cardiac myocyte behavior can be controlled through local 3D microdomains alone. This approach of defining physical cues as independent features may help to advance the elemental design considerations for scaffolds in cardiac tissue engineering and therapeutic microdevices. PMID:20668947

  17. Out-of-plane Block Copolymer Microdomains in High Aspect-Ratio Templates

    NASA Astrophysics Data System (ADS)

    Gadelrab, Karim; Bai, Wubin; Alexander-Katz, Alfredo; Ross, Caroline

    The use of directed self-assembly DSA of block copolymers BCP proved to be a power approach for nanoscale fabrication. It combines the ability of BCPs to self-assemble into nanoscale features with the use of lithographic tools to create controlled long range order. In addition, BCP with highly incompatible blocks (high Flory-Huggins interaction parameter (χ)) offer improvement in resolution, and line edge fluctuations of the self-assembled patterns. Unfortunately, high- χ BCPs usually exhibit large differences in surface affinity between the two blocks, leading to the formation of a surface layer of the lower surface energy block and favoring in-plane orientation of lamellae or cylindrical microdomains. Here, we explore the conditions under which a high χ BCP creates an out-of-plane lamellar structure using functionalized high aspect ratio trenches with preferential walls. We employ the free energy analysis of self-consistent field theory SCFT to identify whether an in-plane or out-of-plane structure is stable for a particular trench width. In addition, we employ the single mode expansion of Ginzburg-Landau free energy expression in the weak segregation limit to analytically construct a phase diagram of the in-plane and out-of-plane lamellae as a function of aspect ratio and surface attraction strength. It is found that achieving an out of plane lamellar structure necessitates a coupling between aspect ratio and surface functionality. In particular, strong side wall attraction results in out-of-plane lamellae when the trench aspect ratio is greater than unity. The results are validated for a lamellar forming polystyrene-block-polydimethylsiloxane (PS-b-PDMS) within trenches made using interference lithography.

  18. Adsorption of phthalic acid esters (PAEs) by amphiphilic polypropylene nonwoven from aqueous solution: the study of hydrophilic and hydrophobic microdomain.

    PubMed

    Zhou, Xiangyu; Wei, Junfu; Zhang, Huan; Liu, Kai; Wang, Han

    2014-05-30

    A kind of amphiphilic polypropylene nonwoven with hydrophilic and hydrophobic microdomain was prepared through electron beam induced graft polymerization and subsequent ring opening reaction and then utilized in the adsorption of phthalic acid esters (PAEs). To elucidate the superiority of such amphiphilic microdomain, a unique structure without hydrophilic part was constructed as comparison. In addition, the adsorption behaviors including adsorption kinetics, isotherms and pH effect were systematically investigated. The result indicated that the amphiphilic structure and the synergy between hydrophilic and hydrophobic microdomain could considerably improve the adsorption capacities, rate and affinity. Particularly the existence of hydrophilic microdomain could reduce the diffusion resistance and energy barrier in the adsorption process. These adsorption results showed that the amphiphilic PP nonwoven have the potential to be used in environmental application. PMID:24721695

  19. Microdomain Formation, Oxidation, and Cation Ordering in LaCa2Fe3O8+y

    DOE PAGESBeta

    Price, Patrick M.; Browning, Nigel D.; Butt, Darryl P.

    2015-03-23

    The compound LaCa2Fe3O8+y, also known as the Grenier phase, is known to undergo an order-disorder transformation (ODT) at high temperatures. Oxidation has been observed when the compound is cooled in air after the ODT. In this study, we have synthesized the Grenier compound in air using traditional solid state reactions and investigated the structure and composition before and after the ODT. Thermal analysis showed that the material undergoes an order-disorder transformation in both oxygen and argon atmospheres with dynamic, temperature dependent, oxidation upon cooling. Results from scanning transmission electron microscopy (STEM) suggest that the Grenier phase has preferential segregation ofmore » Ca and La on the two crystallographic A-sites before the ODT, but a random distribution above the ODT temperature. Furthermore, STEM images suggest the possibility that oxygen excess may exist in La-rich regions within microdomains rather than at microdomain boundaries.« less

  20. Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT

    PubMed Central

    Cremona, M. Laura; Matthies, Heinrich J.G.; Pau, Kelvin; Bowton, Erica; Speed, Nicole; Lute, Brandon J.; Anderson, Monique; Sen, Namita; Robertson, Sabrina D.; Vaughan, Roxanne A.; Rothman, James E.; Galli, Aurelio; Javitch, Jonathan A.; Yamamoto, Ai

    2011-01-01

    Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, following their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We find that the protein Flotillin-1/Reggie-2 (Flot1) is required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we have identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 is also required to localize DAT within plasma membrane microdomains in stable cell lines, and is essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine. PMID:21399631

  1. Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions.

    PubMed

    Danielsen, E Michael; Hansen, Gert H

    2003-10-31

    Epithelial cells that fulfil high-throughput digestive/absorptive functions, such as small intestinal enterocytes and kidney proximal tubule cells, are endowed with a dense apical brush border. It has long been recognized that the microvillar surface of the brush border is organized in cholesterol/sphingolipid-enriched membrane microdomains commonly known as lipid rafts. More recent studies indicate that microvillar rafts, in particular those of enterocytes, have some unusual properties in comparison with rafts present on the surface of other cell types. Thus, microvillar rafts are stable rather than transient/dynamic, and their core components include glycolipids and the divalent lectin galectin-4, which together can be isolated as "superrafts", i.e., membrane microdomains resisting solubilization with Triton X-100 at physiological temperature. These glycolipid/lectin-based rafts serve as platforms for recruitment of GPI-linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol-dependent lipid rafts of a different type from the glycolipid-based rafts at the microvillar surface. The brush border is thus an example of a complex membrane system that harbours at least two different types of lipid raft microdomains, each suited to fulfil specialized functions. This conclusion is in line with an emerging, more varied view of lipid rafts being pluripotent microdomains capable of adapting in size, shape, and content to

  2. Protein profiling of microdomains purified from renal cell carcinoma and normal kidney tissue samples.

    PubMed

    Raimondo, F; Morosi, L; Chinello, C; Perego, R; Bianchi, C; Albo, G; Ferrero, S; Rocco, F; Magni, F; Pitto, M

    2012-04-01

    Renal cell carcinoma (RCC) is representing about 3% of all adult cancers. A promising strategy for cancer biomarker discovery is subcellular comparative proteomics, allowing enriching specific cell compartments and assessing differences in protein expression patterns. We investigated the proteomic profile of a peculiar RCC subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues were analyzed after SDS-PAGE separation by LC-ESI-MS/MS. We identified 93 proteins from MD isolated from RCC tissue, and 98 proteins from ANK MD. About 70% of the identified proteins are membrane-associated and about half of these are known as microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. We chose a panel of proteins to validate their differential expression by WB. In conclusion, our work shows that RCC microdomain proteome is reproducibly different from ANK, and suggests that mining into such differences may support new biomarker discovery. PMID:22159573

  3. Gel-Phase Microdomains and Lipid Rafts in Monolayers Affect the Redox Properties of Ubiquinone-10

    PubMed Central

    Becucci, Lucia; Scaletti, Federica; Guidelli, Rolando

    2011-01-01

    The redox properties of ubiquinone-10 (UQ) were examined in monolayers of mixtures of dioleoylphosphatidylcholine, palmitoylsphingomyelin, and cholesterol of different compositions, self-assembled on a mercury electrode, over the pH range from 7.5 to 9.5. A detailed analysis of the cyclic voltammograms of UQ in the above lipid environments points to a mechanism consisting of an elementary electron transfer step followed by two protonation (or deprotonation) steps in quasiequilibrium and by a further electron transfer step. In a lipid environment of solid-ordered (so) microdomains in a liquid-disordered (ld) matrix, electron transport across the lipid monolayer takes place in the ld phase. In a pure so phase, UQ tends to segregate into UQ-rich pools, exhibiting reversible electron transfer steps. In a lipid environment consisting of liquid-ordered (lo) microdomains (lipid rafts) in an ld matrix, UQ molecules tend to localize along the edge of the lipid rafts. However, in a lipid environment consisting exclusively of lo and so microdomains, UQ molecules tend to segregate into UQ-rich pools. In all lipid environments, electron transport by UQ occurs with the quinone moiety localized on the solution side with respect to the ester linkages of the dioleoylphosphatidylcholine molecules. PMID:21723823

  4. Ca2+ microdomains near plasma membrane Ca2+ channels: impact on cell function.

    PubMed

    Parekh, Anant B

    2008-07-01

    In eukaryotic cells, a rise in cytoplasmic Ca(2+) can activate a plethora of responses that operate on time scales ranging from milliseconds to days. Inherent to the use of a promiscuous signal like Ca(2+) is the problem of specificity: how can Ca(2+) activate some responses but not others? We now know that the spatial profile of the Ca(2+) signal is important Ca(2+) does not simply rise uniformly throughout the cytoplasm upon stimulation but can reach very high levels locally, creating spatial gradients. The most fundamental local Ca(2+) signal is the Ca(2+) microdomain that develops rapidly near open plasmalemmal Ca(2+) channels like voltage-gated L-type (Cav1.2) and store-operated CRAC channels. Recent work has revealed that Ca(2+) microdomains arising from these channels are remarkably versatile in triggering a range of responses that differ enormously in both temporal and spatial profile. Here, I delineate basic features of Ca(2+) microdomains and then describe how these highly local signals are used by Ca(2+)-permeable channels to drive cellular responses. PMID:18467365

  5. Blast exposure causes dynamic microglial/macrophage responses and microdomains of brain microvessel dysfunction.

    PubMed

    Huber, B R; Meabon, J S; Hoffer, Z S; Zhang, J; Hoekstra, J G; Pagulayan, K F; McMillan, P J; Mayer, C L; Banks, W A; Kraemer, B C; Raskind, M A; McGavern, D B; Peskind, E R; Cook, D G

    2016-04-01

    Exposure to blast overpressure (BOP) is associated with behavioral, cognitive, and neuroimaging abnormalities. We investigated the dynamic responses of cortical vasculature and its relation to microglia/macrophage activation in mice using intravital two-photon microscopy following mild blast exposure. We found that blast caused vascular dysfunction evidenced by microdomains of aberrant vascular permeability. Microglial/macrophage activation was specifically associated with these restricted microdomains, as evidenced by rapid microglial process retraction, increased ameboid morphology, and escape of blood-borne Q-dot tracers that were internalized in microglial/macrophage cell bodies and phagosome-like compartments. Microdomains of cortical vascular disruption and microglial/macrophage activation were also associated with aberrant tight junction morphology that was more prominent after repetitive (3×) blast exposure. Repetitive, but not single, BOPs also caused TNFα elevation two weeks post-blast. In addition, following a single BOP we found that aberrantly phosphorylated tau rapidly accumulated in perivascular domains, but cleared within four hours, suggesting it was removed from the perivascular area, degraded, and/or dephosphorylated. Taken together these findings argue that mild blast exposure causes an evolving CNS insult that is initiated by discrete disturbances of vascular function, thereby setting the stage for more protracted and more widespread neuroinflammatory responses. PMID:26777891

  6. Clathrin and Membrane Microdomains Cooperatively Regulate RbohD Dynamics and Activity in Arabidopsis.

    PubMed

    Hao, Huaiqing; Fan, Lusheng; Chen, Tong; Li, Ruili; Li, Xiaojuan; He, Qihua; Botella, Miguel A; Lin, Jinxing

    2014-04-22

    Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin- and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways. PMID:24755455

  7. LOCALIZATION AND PROTEOMIC CHARACTERIZATION OF CHOLESTEROL-RICH MEMBRANE MICRODOMAINS IN THE INNER EAR

    PubMed Central

    Thomas, Paul V.; Cheng, Andrew L.; Colby, Candice C.; Liu, Liqian; Patel, Chintan K.; Josephs, Lydia; Duncan, R. Keith

    2014-01-01

    Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein-protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology. PMID:24713161

  8. Eye lens membrane junctional microdomains: a comparison between healthy and pathological cases

    NASA Astrophysics Data System (ADS)

    Buzhynskyy, Nikolay; Sens, Pierre; Behar-Cohen, Francine; Scheuring, Simon

    2011-08-01

    The eye lens is a transparent tissue constituted of tightly packed fiber cells. To maintain homeostasis and transparency of the lens, the circulation of water, ions and metabolites is required. Junctional microdomains connect the lens cells and ensure both tight cell-to-cell adhesion and intercellular flow of fluids through a microcirculation system. Here, we overview membrane morphology and tissue functional requirements of the mammalian lens. Atomic force microscopy (AFM) has opened up the possibility of visualizing the junctional microdomains at unprecedented submolecular resolution, revealing the supramolecular assembly of lens-specific aquaporin-0 (AQP0) and connexins (Cx). We compare the membrane protein assembly in healthy lenses with senile and diabetes-II cataract cases and novel data of the lens membranes from a congenital cataract. In the healthy case, AQP0s form characteristic square arrays confined by connexons. In the cases of senile and diabetes-II cataract patients, connexons were degraded, leading to malformation of AQP0 arrays and breakdown of the microcirculation system. In the congenital cataract, connexons are present, indicating probable non-membranous grounds for lens opacification. Further, we discuss the energetic aspects of the membrane organization in junctional microdomains. The AFM hence becomes a biomedical nano-imaging tool for the analysis of single-membrane protein supramolecular association in healthy and pathological membranes.

  9. Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

    PubMed

    Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

    2016-08-15

    Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. PMID:27375200

  10. Arabidopsis mutants in sphingolipid synthesis as tools to understand the structure and function of membrane microdomains in plasmodesmata

    PubMed Central

    González-Solís, Ariadna; Cano-Ramírez, Dora L.; Morales-Cedillo, Francisco; Tapia de Aquino, Cinthya; Gavilanes-Ruiz, Marina

    2013-01-01

    Plasmodesmata—intercellular channels that communicate adjacent cells—possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures. PMID:24478783

  11. FRET-Based Nanobiosensors for Imaging Intracellular Ca²⁺ and H⁺ Microdomains.

    PubMed

    Zamaleeva, Alsu I; Despras, Guillaume; Luccardini, Camilla; Collot, Mayeul; de Waard, Michel; Oheim, Martin; Mallet, Jean-Maurice; Feltz, Anne

    2015-01-01

    Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca(2+) and H⁺ transients. Our sensors combine a commercially available CANdot(®)565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca(2+) or H⁺ probes. These 'Rubies' are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) for H⁺ or Ca(2+) sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca(2+) and pH transients using live-cell fluorescence imaging. PMID:26404317

  12. Orientation of Microdomains of Block Copolymers by Zone casting

    NASA Astrophysics Data System (ADS)

    Tang, Chuanbing; Matyjaszewski, Krzysztof; Kowalewski, Tomasz

    2007-03-01

    As a ``bottom up'' method, the self-assembly of block copolymers plays a vital role in the development of soft lithography for the fabrication of microelectronic devices. A variety of methods have been developed toward better and more precise controlled patterns on solid substrates. This presentation will describe a novel solution casting technique, ``zone casting'', to induce orientation of cylindrical and lamellar microphase-separated domains of various block copolymers.

  13. Transition from Nanodomains to Microdomains Induced by Exposure of Lipid Monolayers to Air

    PubMed Central

    Coban, Oana; Popov, Jesse; Burger, Melanie; Vobornik, Dusan; Johnston, Linda J.

    2007-01-01

    The morphology of monolayers prepared from ternary lipid mixtures that have coexisting fluid phases has been examined by atomic force microscopy for samples transferred to mica before and after exposure to air. Mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol with either egg sphingomyelin or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied at several surface pressures. Both lipid mixtures have a combination of small islands and large microdomains at low surface pressure (5–10 mN/m) for monolayers deposited in either air or nitrogen. By contrast, monolayers have small interconnected nanodomains when deposited under nitrogen at 30 mN/m but mixtures of large microdomains and small nanodomains when transferred after exposure to air. These results are consistent with an earlier report that concluded that the formation of large domains at high surface pressures (>30 mN/m) for monolayers exposed to air is caused by lipid oxidation. However, the higher spatial resolution available with atomic force microscopy indicates that exposure of the monolayers to air leads to an increase in the size of preexisting nanodomains, rather than a change in the miscibility pressure. Examination of changes in surface morphology as a function of surface pressure demonstrate a gradual evolution in size and surface coverage for both nano- and microdomains, before formation of a network of interconnected nanodomains. Similar studies for binary mixtures in the absence of cholesterol indicate that lipid oxidation results in analogous changes in domain size for monolayers with coexisting gel and fluid phases. These results illustrate the importance of using techniques capable of probing the nanoscale organization of membranes. PMID:17237193

  14. Overexpression of BAX INHIBITOR-1 Links Plasma Membrane Microdomain Proteins to Stress1[OPEN

    PubMed Central

    Ishikawa, Toshiki; Aki, Toshihiko; Yanagisawa, Shuichi; Uchimiya, Hirofumi; Kawai-Yamada, Maki

    2015-01-01

    BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death. PMID:26297139

  15. Magnetic alignment of block copolymer microdomains by intrinsic chain anisotropy

    DOE PAGESBeta

    Rokhlenko, Yekaterina; Yager, Kevin G.; Gopinadhan, Manesh; Osuji, Chinedum O.; Zhang, Kai; O'Hern, Corey S.; Larson, Steven R.; Gopalan, Padma; Majewski, Pawel W.

    2015-12-18

    We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δχ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δχ ≈ 2×10–8. From field-dependent scattering data, we estimate that grains of ≈ 1.2 μm are present during alignment. Furthermore, these results demonstrate that intrinsic anisotropymore » is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.« less

  16. Magnetic Alignment of Block Copolymer Microdomains by Intrinsic Chain Anisotropy

    NASA Astrophysics Data System (ADS)

    Rokhlenko, Yekaterina; Gopinadhan, Manesh; Osuji, Chinedum O.; Zhang, Kai; O'Hern, Corey S.; Larson, Steven R.; Gopalan, Padma; Majewski, Paweł W.; Yager, Kevin G.

    2015-12-01

    We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δ χ , that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δ χ ≈2 ×1 0-8. From field-dependent scattering data, we estimate that grains of ≈1.2 μ m are present during alignment. These results demonstrate that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.

  17. Magnetic alignment of block copolymer microdomains by intrinsic chain anisotropy

    SciTech Connect

    Rokhlenko, Yekaterina; Yager, Kevin G.; Gopinadhan, Manesh; Osuji, Chinedum O.; Zhang, Kai; O'Hern, Corey S.; Larson, Steven R.; Gopalan, Padma; Majewski, Pawel W.

    2015-12-18

    We examine the role of intrinsic chain susceptibility anisotropy in magnetic field directed self-assembly of a block copolymer using in situ x-ray scattering. Alignment of a lamellar mesophase is observed on cooling across the disorder-order transition with the resulting orientational order inversely proportional to the cooling rate. We discuss the origin of the susceptibility anisotropy, Δχ, that drives alignment and calculate its magnitude using coarse-grained molecular dynamics to sample conformations of surface-tethered chains, finding Δχ ≈ 2×10–8. From field-dependent scattering data, we estimate that grains of ≈ 1.2 μm are present during alignment. Furthermore, these results demonstrate that intrinsic anisotropy is sufficient to support strong field-induced mesophase alignment and suggest a versatile strategy for field control of orientational order in block copolymers.

  18. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    PubMed Central

    Brügger, Britta; Krautkrämer, Ellen; Tibroni, Nadine; Munte, Claudia E; Rauch, Susanne; Leibrecht, Iris; Glass, Bärbel; Breuer, Sebastian; Geyer, Matthias; Kräusslich, Hans-Georg; Kalbitzer, Hans Robert; Wieland, Felix T; Fackler, Oliver T

    2007-01-01

    Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo. PMID:17908312

  19. Microdomains shift and rotate in the lateral wall of cochlear outer hair cells.

    PubMed

    Kitani, Rei; Park, Channy; Kalinec, Federico

    2013-01-01

    Outer hair cell (OHC) electromotility, a response consisting of reversible changes in cell length and diameter induced by electrical stimulation, confers remarkable sensitivity and frequency resolution to the mammalian inner ear. Looking for a better understanding of this mechanism, we labeled isolated guinea pig OHCs with microspheres and, using high-speed video recording, investigated their movements at the apical, mid, and basal regions of osmotically and electrically stimulated cells. After hypoosmotic challenge, OHCs shortened and their diameter increased, with microspheres moving always toward the central plane; iso-osmolarity returned OHCs to their original shape and microspheres to their original positions. Under electrical stimulation, microspheres exhibited robust movements, with their displacement vectors changing in direction from random to parallel to the longitudinal axis of the cells with peak reorientation speeds of up to 6 rad/s and returning to random after 5 min without stimulation. Alterations in plasma-membrane cholesterol levels as well as cytoskeleton integrity affected microsphere responses. We concluded that microspheres attach to different molecular microdomains, and these microdomains are able to shift and rotate in the plane of the OHC lateral wall with a dynamics tightly regulated by membrane lipid composition and the cortical cytoskeleton. PMID:23332054

  20. The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription

    PubMed Central

    Shimi, Takeshi; Pfleghaar, Katrin; Kojima, Shin-ichiro; Pack, Chan-Gi; Solovei, Irina; Goldman, Anne E.; Adam, Stephen A.; Shumaker, Dale K.; Kinjo, Masataka; Cremer, Thomas; Goldman, Robert D.

    2008-01-01

    The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH) analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and immunofluorescence localization of modified histones demonstrate that gene-rich euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated RNA polymerase II (Pol II) and histone marks for active transcription suggest that blebs are transcriptionally active. However, in vivo labeling of RNA indicates that transcription is decreased, suggesting that the LA/C-rich microenvironment induces promoter proximal stalling of Pol II. We propose that different lamins are organized into separate, but interacting, microdomains and that LB1 is essential for their organization. Our evidence suggests that the organization and regulation of chromatin are influenced by interconnections between these lamin microdomains. PMID:19141474

  1. STIM1-dependent Ca2+ microdomains are required for myofilament remodeling and signaling in the heart

    PubMed Central

    Parks, Cory; Alam, Mohammad Afaque; Sullivan, Ryan; Mancarella, Salvatore

    2016-01-01

    In non-excitable cells stromal interaction molecule 1 (STIM1) is a key element in the generation of Ca2+ signals that lead to gene expression, migration and cell proliferation. A growing body of literature suggests that STIM1 plays a key role in the development of pathological cardiac hypertrophy. However, the precise mechanisms involving STIM-dependent Ca2+ signaling in the heart are not clearly established. Here, we have investigated the STIM1-associated Ca2+ signals in cardiomyocytes and their relevance to pathological cardiac remodeling. We show that mice with inducible, cardiac-restricted, ablation of STIM1 exhibited left ventricular reduced contractility, which was corroborated by impaired single cell contractility. The spatial properties of STIM1-dependent Ca2+ signals determine restricted Ca2+ microdomains that regulate myofilament remodeling and activate spatially segregated pro-hypertrophic factors. Indeed, mice lacking STIM1 showed less adverse structural remodeling in response to pressure overload-induced cardiac hypertrophy. These results highlight how STIM1-dependent Ca2+ microdomains have a major impact on intracellular Ca2+ homeostasis, cytoskeletal remodeling and cellular signaling, even when excitation-contraction coupling is present. PMID:27150728

  2. Monte Carlo simulation of the effects of vesicle geometry on calcium microdomains and neurotransmitter release

    NASA Astrophysics Data System (ADS)

    Limsakul, Praopim; Modchang, Charin

    2016-07-01

    We investigate the effects of synaptic vesicle geometry on Ca2+ diffusion dynamics in presynaptic terminals using MCell, a realistic Monte Carlo algorithm that tracks individual molecules. By modeling the vesicle as a sphere and an oblate or a prolate spheroid with a reflective boundary, we measure the Ca2+ concentration at various positions relative to the vesicle. We find that the presence of a vesicle as a diffusion barrier modifies the shape of the [Ca2+] microdomain in the vicinity of the vesicle. Ca2+ diffusion dynamics also depend on the distance between the vesicle and the voltage-gated calcium channels (VGCCs) and on the shape of the vesicle. The oblate spheroidal vesicle increases the [Ca2+] up to six times higher than that in the absence of a vesicle, while the prolate spheroidal vesicle can increase the [Ca2+] only 1.4 times. Our results also show that the presence of vesicles that have different geometries can maximally influence the [Ca2+] microdomain when the vesicle is located less than 50 nm from VGCCs.

  3. Extrapolating microdomain Ca2+ dynamics using BK channels as a Ca2+ sensor

    PubMed Central

    Hou, Panpan; Xiao, Feng; Liu, Haowen; Yuchi, Ming; Zhang, Guohui; Wu, Ying; Wang, Wei; Zeng, Wenping; Ding, Mingyue; Cui, Jianming; Wu, Zhengxing; Wang, Lu-Yang; Ding, Jiuping

    2016-01-01

    Ca2+ ions play crucial roles in mediating physiological and pathophysiological processes, yet Ca2+ dynamics local to the Ca2+ source, either from influx via calcium permeable ion channels on plasmic membrane or release from internal Ca2+ stores, is difficult to delineate. Large-conductance calcium-activated K+ (BK-type) channels, abundantly distribute in excitable cells and often localize to the proximity of voltage-gated Ca2+ channels (VGCCs), spatially enabling the coupling of the intracellular Ca2+ signal to the channel gating to regulate membrane excitability and spike firing patterns. Here we utilized the sensitivity and dynamic range of BK to explore non-uniform Ca2+ local transients in the microdomain of VGCCs. Accordingly, we applied flash photolysis of caged Ca2+ to activate BK channels and determine their intrinsic sensitivity to Ca2+. We found that uncaging Ca2+ activated biphasic BK currents with fast and slow components (time constants being τf ≈ 0.2 ms and τs ≈ 10 ms), which can be accounted for by biphasic Ca2+ transients following light photolysis. We estimated the Ca2+-binding rate constant kb (≈1.8 × 108 M−1s−1) for mSlo1 and further developed a model in which BK channels act as a calcium sensor capable of quantitatively predicting local microdomain Ca2+ transients in the vicinity of VGCCs during action potentials. PMID:26776352

  4. Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

    SciTech Connect

    Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

    2008-01-15

    The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped viruses indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.

  5. Extrapolating microdomain Ca(2+) dynamics using BK channels as a Ca(2+) sensor.

    PubMed

    Hou, Panpan; Xiao, Feng; Liu, Haowen; Yuchi, Ming; Zhang, Guohui; Wu, Ying; Wang, Wei; Zeng, Wenping; Ding, Mingyue; Cui, Jianming; Wu, Zhengxing; Wang, Lu-Yang; Ding, Jiuping

    2016-01-01

    Ca(2+) ions play crucial roles in mediating physiological and pathophysiological processes, yet Ca(2+) dynamics local to the Ca(2+) source, either from influx via calcium permeable ion channels on plasmic membrane or release from internal Ca(2+) stores, is difficult to delineate. Large-conductance calcium-activated K(+) (BK-type) channels, abundantly distribute in excitable cells and often localize to the proximity of voltage-gated Ca(2+) channels (VGCCs), spatially enabling the coupling of the intracellular Ca(2+) signal to the channel gating to regulate membrane excitability and spike firing patterns. Here we utilized the sensitivity and dynamic range of BK to explore non-uniform Ca(2+) local transients in the microdomain of VGCCs. Accordingly, we applied flash photolysis of caged Ca(2+) to activate BK channels and determine their intrinsic sensitivity to Ca(2+). We found that uncaging Ca(2+) activated biphasic BK currents with fast and slow components (time constants being τf ≈ 0.2 ms and τs ≈ 10 ms), which can be accounted for by biphasic Ca(2+) transients following light photolysis. We estimated the Ca(2+)-binding rate constant kb (≈1.8 × 10(8)  M(-1) s(-1)) for mSlo1 and further developed a model in which BK channels act as a calcium sensor capable of quantitatively predicting local microdomain Ca(2+) transients in the vicinity of VGCCs during action potentials. PMID:26776352

  6. A host cell membrane microdomain is a critical factor for organelle discharge by Toxoplasma gondii.

    PubMed

    Tahara, Michiru; Andrabi, Syed Bilal Ahmad; Matsubara, Ryuma; Aonuma, Hiroka; Nagamune, Kisaburo

    2016-10-01

    Host cell microdomains are involved in the attachment, entry, and replication of intracellular microbial pathogens. Entry into the host cell of Toxoplasma gondii and the subsequent survival of this protozoan parasite are tightly coupled with the proteins secreted from organelle called rhoptry. The rhoptry proteins are rapidly discharged into clusters of vesicles, called evacuoles, which are then delivered to parasitophorous vacuoles (PVs) or nucleus. In this study, we examined the roles of two host cell microdomain components, cholesterol and glycosylphosphatidylinositol (GPI), in evacuole formation. The acute depletion of cholesterol from the host cell plasma membrane blocked evacuole formation but not invasion. Whereas the lack of host cell GPI also altered evacuole formation but not invasion, instead inducing excess evacuole formation. The latter effect was not influenced by the evacuole-inhibiting effects of host cell cholesterol depletion, indicating the independent roles of host GPI and cholesterol in evacuole formation. In addition, the excess formation of evacuoles resulted in the enhanced recruitment of host mitochondria and endoplasmic reticulum to PVs, which in turn stimulated the growth of the parasite. PMID:27217289

  7. Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana[C][W

    PubMed Central

    Jarsch, Iris K.; Konrad, Sebastian S.A.; Stratil, Thomas F.; Urbanus, Susan L.; Szymanski, Witold; Braun, Pascal; Braun, Karl-Heinz; Ott, Thomas

    2014-01-01

    Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo. PMID:24714763

  8. Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling1

    PubMed Central

    Fessler, Michael B.; Parks, John S.

    2011-01-01

    Lipid rafts and caveolae play a pivotal role in organization of signaling by Toll-like Receptor (TLR)4 and several other immune receptors. Beyond the simple cataloguing of signaling events compartmentalized by these membrane microdomains, recent studies have revealed the surprisingly central importance of dynamic remodeling of membrane lipid domains to immune signaling. Simple interventions upon membrane lipid, such as changes in cholesterol loading or crosslinking of raft lipids, are sufficient to induce micron-scale reordering of membranes and their protein cargo with consequent signal transduction. In this review, using TLR signaling in the macrophage as a central focus, we discuss emerging evidence that environmental and genetic perturbations of membrane lipid regulate protein signaling, illustrate how homeostatic flow of cholesterol and other lipids through rafts regulates the innate immune response, and highlight recent attempts to harness these insights towards therapeutic development. PMID:21810617

  9. Simulations Show that Virus Assembly and Budding Are Facilitated by Membrane Microdomains

    PubMed Central

    Ruiz-Herrero, Teresa; Hagan, Michael F.

    2015-01-01

    For many viruses, assembly and budding occur simultaneously during virion formation. Understanding the mechanisms underlying this process could promote biomedical efforts to block viral propagation and enable use of capsids in nanomaterials applications. To this end, we have performed molecular dynamics simulations on a coarse-grained model that describes virus assembly on a fluctuating lipid membrane. Our simulations show that the membrane can promote association of adsorbed subunits through dimensional reduction, but it also introduces thermodynamic and kinetic effects that can inhibit complete assembly. We find several mechanisms by which membrane microdomains, such as lipid rafts, reduce these effects, and thus, enhance assembly. We show how these predicted mechanisms can be experimentally tested. Furthermore, the simulations demonstrate that assembly and budding depend crucially on the system dynamics via multiple timescales related to membrane deformation, protein diffusion, association, and adsorption onto the membrane. PMID:25650926

  10. Adult neural stem cells in distinct microdomains generate previously unknown interneuron types

    PubMed Central

    Merkle, Florian T.; Fuentealba, Luis C.; Sanders, Timothy A.; Magno, Lorenza; Kessaris, Nicoletta; Alvarez-Buylla, Arturo

    2014-01-01

    Throughout life, neural stem cells (NSCs) in different domains of the ventricular-subventricular zone (V-SVZ) of the adult rodent brain generate several subtypes of interneurons that regulate the function of the olfactory bulb (OB). The full extent of diversity among adult NSCs and their progeny is not known. Here, we report the generation of at least four previously unknown OB interneuron subtypes that are produced in finely patterned progenitor domains in the anterior ventral V-SVZ of both the neonatal and adult brain. Progenitors of these novel interneurons are responsive to sonic hedgehog (SHH) and are organized into microdomains that correlate with the expression domains of the Nkx6.2 and Zic family of transcription factors. This work reveals an unexpected degree of complexity in the specification and patterning of NSCs in the postnatal mouse brain. PMID:24362763

  11. Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

    PubMed

    Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

    2016-01-01

    Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes. PMID:27207290

  12. Determination of seizure propagation across microdomains using spectral measures of causality.

    PubMed

    Basu, Ishita; Kudela, Pawel; Anderson, William S

    2014-01-01

    The use of microelectrode arrays to measure electrical activity from the surface of the brain is increasingly being investigated as a means to improve seizure focus localization. In this work, we determine seizure propagation across microdomains sampled by such microelectrode arrays and compare the results using two widely used frequency domain measures of causality, namely the partial directed coherence and the directed direct transfer function. We show that these two measures produce very similar propagation patterns for simulated microelectrode activity over a relatively smaller number of channels. However as the number of channels increases, partial directed coherence produces better estimates of the actual propagation pattern. Additionally, we apply these two measures to determine seizure propagation over microelectrode arrays measured from a patient undergoing intracranial monitoring for seizure focus localization and find very similar patterns which also agree with a threshold based reconstruction during seizure onset. PMID:25571448

  13. Diffusing Polymers in Confined Microdomains and Estimation of Chromosomal Territory Sizes from Chromosome Capture Data

    NASA Astrophysics Data System (ADS)

    Amitai, A.; Holcman, D.

    2013-06-01

    Is it possible to extract the size and structure of chromosomal territories (confined domain) from the encounter frequencies of chromosomal loci? To answer this question, we estimate the mean time for two monomers located on the same polymer to encounter, which we call the mean first encounter time in a confined microdomain (MFETC). We approximate the confined domain geometry by a harmonic potential well and obtain an asymptotic expression that agrees with Brownian simulations for the MFETC as a function of the polymer length, the radius of the confined domain, and the activation distance radius ɛ at which the two searching monomers meet. We illustrate the present approach using chromosome capture data for the encounter rate distribution of two loci depending on their distances along the DNA. We estimate the domain size that restricts the motion of one of these loci for chromosome II in yeast.

  14. MAL Is a Regulator of the Recruitment of Myelin Protein PLP to Membrane Microdomains

    PubMed Central

    Bijlard, Marjolein; de Jonge, Jenny C.; Klunder, Bert; Nomden, Anita; Hoekstra, Dick; Baron, Wia

    2016-01-01

    In oligodendrocytes (OLGs), an indirect, transcytotic pathway is mediating transport of de novo synthesized PLP, a major myelin specific protein, from the apical-like plasma membrane to the specialized basolateral-like myelin membrane to prevent its premature compaction. MAL is a well-known regulator of polarized trafficking in epithelial cells, and given its presence in OLGs it was therefore of interest to investigate whether MAL played a similar role in PLP transport in OLGs, taking into account its timely expression in these cells. Our data revealed that premature expression of mCherry-MAL in oligodendrocyte progenitor cells interfered with terminal OLG differentiation, although myelin membrane formation per se was not impaired. In fact, also PLP transport to myelin membranes via the cell body plasma membrane was unaffected. However, the typical shift of PLP from TX-100-insoluble membrane domains to CHAPS-resistant, but TX-100-soluble membrane domains, seen in the absence of MAL expression, is substantially reduced upon expression of the MAL protein. Interestingly, not only in vitro, but also in developing brain a strongly diminished shift from TX-100 resistant to TX-100 soluble domains was observed. Consistently, the MAL-expression mediated annihilation of the typical membrane microdomain shift of PLP is also reflected by a loss of the characteristic surface expression profile of conformation-sensitive anti-PLP antibodies. Hence, these findings suggest that MAL is not involved in vesicular PLP trafficking to either the plasma membrane and/or the myelin membrane as such. Rather, we propose that MAL may regulate PLP’s distribution into distinct membrane microdomains that allow for lateral diffusion of PLP, directly from the plasma membrane to the myelin membrane once the myelin sheath has been assembled. PMID:27171274

  15. The sensing of membrane microdomains based on pore-forming toxins.

    PubMed

    Skočaj, M; Bakrač, B; Križaj, I; Maček, P; Anderluh, G; Sepčić, K

    2013-01-01

    Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside G(M1), and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Nontoxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains. PMID:23244522

  16. Intriguing transmission electron microscopy images observed for perpendicularly oriented cylindrical microdomains of block copolymers

    NASA Astrophysics Data System (ADS)

    Ohnogi, Hiroshi; Isshiki, Toshiyuki; Sasaki, Sono; Sakurai, Shinichi

    2014-08-01

    Intriguing images of dislocation structures were observed by the transmission electron microscopy (TEM) technique for hexagonally packed cylindrical microdomains in a block copolymer (polystyrene-block-polyethylenebutylene-block-polystyrene triblock copolymer) film. The polystyrene (PS) cylinders were embedded in the polyethylenebutylene (PEB) matrix and oriented perpendicular to the surface of the thin section for the TEM observations. In order to understand such strange dislocation structures, we applied an image processing technique using two-dimensional Fourier transform (FT) and inverse Fourier transform (IFT) methods. It was found that these intriguing images were not ascribed to real dislocation structures but were fake ones due to the moiré effect caused by the overlapping of hexagons with a slightly mismatched orientation. Furthermore, grain boundaries in the ultrathin section can be identified by image processing using FT and IFT methods.Intriguing images of dislocation structures were observed by the transmission electron microscopy (TEM) technique for hexagonally packed cylindrical microdomains in a block copolymer (polystyrene-block-polyethylenebutylene-block-polystyrene triblock copolymer) film. The polystyrene (PS) cylinders were embedded in the polyethylenebutylene (PEB) matrix and oriented perpendicular to the surface of the thin section for the TEM observations. In order to understand such strange dislocation structures, we applied an image processing technique using two-dimensional Fourier transform (FT) and inverse Fourier transform (IFT) methods. It was found that these intriguing images were not ascribed to real dislocation structures but were fake ones due to the moiré effect caused by the overlapping of hexagons with a slightly mismatched orientation. Furthermore, grain boundaries in the ultrathin section can be identified by image processing using FT and IFT methods. Electronic supplementary information (ESI) available. See DOI: 10.1039/c

  17. Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains

    PubMed Central

    SHERPA, ANG DOMA; VAN DE NES, PAULA; XIAO, FANRONG; WEEDON, JEREMY; HRABETOVA, SABINA

    2014-01-01

    One of the hallmarks of numerous life-threatening and debilitating brain diseases is cellular swelling that negatively impacts extracellular space (ECS) structure. The ECS structure is determined by two macroscopic parameters, namely tortuosity (λ) and volume fraction (α). Tortuosity represents hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium. Volume fraction is the proportion of tissue volume occupied by the ECS. From a clinical perspective, it is essential to recognize which factors determine the ECS parameters and how these factors change in brain diseases. Previous studies demonstrated that dead-space (DS) microdomains increased λ during ischemia and hypotonic stress, as these pocket-like structures transiently trapped diffusing molecules. We hypothesize that astrocytes play a key role in the formation of DS microdomains because their thin processes have concave shapes that may elongate as astrocytes swell in these pathologies. Here we selectively swelled astrocytes in the somatosensory neocortex of rat brain slices with a gliotoxin DL-α-Aminoadipic Acid (DL-AA), and we quantified the ECS parameters using Integrative Optical Imaging (IOI) and Real-Time Iontophoretic (RTI) diffusion methods. We found that α decreased and λ increased during DL-AA application. During recovery, α was restored whereas λ remained elevated. Increase in λ during astrocytic swelling and recovery is consistent with the formation of DS microdomains. Our data attribute to the astrocytes an important role in determining the ECS parameters, and indicate that extracellular diffusion can be improved not only by reducing the swelling but also by disrupting the DS microdomains. PMID:24687699

  18. Deep-apical tubules: dynamic lipid-raft microdomains in the brush-border region of enterocytes.

    PubMed

    Hansen, Gert H; Pedersen, Jens; Niels-Christiansen, Lise-Lotte; Immerdal, Lissi; Danielsen, E Michael

    2003-07-01

    The brush border of small intestinal enterocytes is highly enriched in cholesterol- and glycosphingolipid-containing membrane microdomains, commonly termed as lipid 'rafts'. Functionally, transcytosis of IgA and exocytosis of newly made brush-border proteins in enterocytes occur through apical lipid raft-containing compartments, but little is otherwise known about these raft microdomains. We therefore studied in closer detail apical lipid-raft compartments in enterocytes by immunogold electron microscopy and biochemical analyses. Novel membrane structures, deep-apical tubules, were visualized by the non-permeable surface marker Ruthenium Red in the brush-border region of the cells. The surface-connected tubules were labelled by antibodies to caveolin-1 and the glycolipid asialo G(M1), and they were sensitive to cholesterol depletion by methyl-beta-cyclodextrin, indicating the presence of raft microdomains. Deep-apical tubules were positioned close to the actin rootlets of adjacent microvilli in the terminal web region, which had a diameter of 50-100 nm, and penetrated up to 1 microm into the cytoplasm. Markers for transcytosis, IgA and the polymeric immunoglobulin receptor, as well as the resident brush-border enzyme aminopeptidase N, were present in these deep-apical tubules. We propose that deep-apical tubules are a specialized lipid-raft microdomain in the brush-border region functioning as a hub in membrane trafficking at the brush border. In addition, the sensitivity to cholesterol depletion suggests that deep-apical tubules function as a cell-surface membrane reservoir for cholesterol and for rapid adaptive changes in the size of microvilli at the brush border. PMID:12689332

  19. Platelet activating factor-induced ceramide micro-domains drive endothelial NOS activation and contribute to barrier dysfunction.

    PubMed

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  20. Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

    PubMed Central

    Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

    2013-01-01

    The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

  1. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease.

    PubMed

    Sprenger, Julia U; Perera, Ruwan K; Steinbrecher, Julia H; Lehnart, Stephan E; Maier, Lars S; Hasenfuss, Gerd; Nikolaev, Viacheslav O

    2015-01-01

    3',5'-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced β-adrenergic receptor-cAMP signalling to SERCA. PMID:25917898

  2. Epstein-Barr virus LMP1 modulates lipid raft microdomains and the vimentin cytoskeleton for signal transduction and transformation.

    PubMed

    Meckes, David G; Menaker, Nathan F; Raab-Traub, Nancy

    2013-02-01

    The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction. PMID:23152522

  3. Coronavirus and Influenza Virus Proteolytic Priming Takes Place in Tetraspanin-Enriched Membrane Microdomains

    PubMed Central

    Earnest, James T.; Hantak, Michael P.; Park, Jung-Eun

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. IMPORTANCE Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular

  4. Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity

    PubMed Central

    Mirzapoiazova, Tamara; Lennon, Frances E.; Mambetsariev, Bolot; Allen, Michael; Riehm, Jacob; Poroyko, Valeriy A.; Singleton, Patrick A.

    2015-01-01

    Defects in vascular integrity are an initiating factor in several disease processes. We have previously reported that high molecular weight hyaluronan (HMW-HA), a major glycosaminoglycan in the body, promotes rapid signal transduction in human pulmonary microvascular endothelial cells (HPMVEC) leading to barrier enhancement. In contrast, low molecular weight hyaluronan (LMW-HA), produced in disease states by hyaluronidases and reactive oxygen species (ROS), induces HPMVEC barrier disruption. However, the mechanism(s) of sustained barrier regulation by HA are poorly defined. Our results indicate that long-term (6–24 hours) exposure of HMW-HA induced release of a novel type of extracellular vesicle from HLMVEC called enlargeosomes (characterized by AHNAK expression) while LMW-HA long-term exposure promoted release of exosomes (characterized by CD9, CD63, and CD81 expression). These effects were blocked by inhibiting caveolin-enriched microdomain (CEM) formation. Further, inhibiting enlargeosome release by annexin II siRNA attenuated the sustained barrier enhancing effects of HMW-HA. Finally, exposure of isolated enlargeosomes to HPMVEC monolayers generated barrier enhancement while exosomes led to barrier disruption. Taken together, these results suggest that differential release of extracellular vesicles from CEM modulate the sustained HPMVEC barrier regulation by HMW-HA and LMW-HA. HMW-HA-induced specialized enlargeosomes can be a potential therapeutic strategy for diseases involving impaired vascular integrity. PMID:26447809

  5. Tetraspanin 8 is an interactor of the metalloprotease meprin β within tetraspanin-enriched microdomains.

    PubMed

    Schmidt, Frederike; Müller, Miryam; Prox, Johannes; Arnold, Philipp; Schönherr, Caroline; Tredup, Claudia; Minder, Petra; Ebsen, Henriette; Janssen, Ottmar; Annaert, Wim; Pietrzik, Claus; Schmidt-Arras, Dirk; Sterchi, Erwin E; Becker-Pauly, Christoph

    2016-09-01

    Meprin β is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin β cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid β peptides and implicating a role of meprin β in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin β, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin β. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin β. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin β. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin β nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin β at the cell surface with impact on certain proteolytic processes that have to be further identified. PMID:27180358

  6. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

    PubMed

    Ryu, Yong-Sang; Wittenberg, Nathan J; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  7. HTLV-1 Tax deregulates autophagy by recruiting autophagic molecules into lipid raft microdomains

    PubMed Central

    Ren, Tong; Takahashi, Yoshinori; Liu, Xin; Loughran, Thomas P.; Sun, Shao-Cong; Wang, Hong-Gang; Cheng, Hua

    2014-01-01

    The retroviral oncoprotein Tax from Human T cell leukemia virus type 1 (HTLV-1), an etiological factor that causes adult T cell leukemia and lymphoma, plays a crucial role in initiating T lymphocyte transformation by inducing oncogenic signaling activation. We here report that Tax is a determining factor for dysregulation of autophagy in HTLV-1-transformed T cells and Tax-immortalized CD4 memory T cells. Tax facilitated autophagic process by activating IκB kinase complex, which subsequently recruited an autophagy molecular complex containing Beclin1 and Bif-1 to the lipid raft microdomains. Tax engaged a crosstalk between IκB kinase complex and autophagic molecule complex by directly interacting with both complexes, promoting assembly of LC3+ autophagosomes. Moreover, expression of lipid raft-targeted Bif-1 or Beclin1 was sufficient to induce formation of LC3+ autophagosomes, suggesting that Tax recruitment of autophagic molecules to lipid rafts is a dominant strategy to deregulate autophagy in the context of HTLV-1 transformation of T cells. Furthermore, depletion of autophagy molecules such as Beclin1 and PI3 kinase class III resulted in impaired growth of HTLV-1-transformed T cells, indicating a critical role of Tax-deregulated autophagy in promoting survival and transformation of virally infected T cells. PMID:24362528

  8. Out-of-plane Block Copolymer Microdomains in High Aspect-Ratio Templates

    NASA Astrophysics Data System (ADS)

    Gadelrab, Karim; Bai, Wubin; Alexander-Katz, Alfredo; Ross, Caroline

    Directed self-assembly DSA of block copolymers BCP proved to be a power approach for nanoscale fabrication. In addition, BCP with highly incompatible blocks (high Flory-Huggins interaction parameter (χ)) offer improvement in resolution of the BCP patterns. Unfortunately, high- χ BCPs usually exhibit large differences in surface affinity between the two blocks, forming a surface layer of the lower surface energy block and favoring in-plane orientation of lamellae or cylindrical microdomains. Here, we explore the conditions under which a high χ BCP creates an out-of-plane lamellar structure using high aspect ratio trenches with preferential walls. We employ self-consistent field theory SCFT and single mode expansion of Ginzburg-Landau free energy expression in the weak segregation limit to analytically construct a phase diagram of the in- and out-of-plane lamellae as a function of aspect ratio and surface affinity. It is found that achieving an out of plane lamellar structure necessitates a coupling between aspect ratio and surface functionality. In particular, strong side wall attraction results in out-of-plane lamellae when the trench aspect ratio is greater than unity. The results are validated for a polystyrene-block-polydimethylsiloxane (PS-b-PDMS) system within trenches made using interference lithography.

  9. Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

    DOE PAGESBeta

    Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; Lee, Sang -Wook; Sohn, Youngjoo; Oh, Sang -Hyun; Parikh, Atul N.; Lee, Sin -Doo

    2016-05-27

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed betweenmore » the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Furthermore, our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates.« less

  10. The Intracellular Interactome of Tetraspanin-enriched Microdomains Reveals Their Function as Sorting Machineries toward Exosomes*

    PubMed Central

    Perez-Hernandez, Daniel; Gutiérrez-Vázquez, Cristina; Jorge, Inmaculada; López-Martín, Soraya; Ursa, Angeles; Sánchez-Madrid, Francisco; Vázquez, Jesús; Yáñez-Mó, María

    2013-01-01

    Extracellular vesicles are emerging as a potent mechanism of intercellular communication because they can systemically exchange genetic and protein material between cells. Tetraspanin molecules are commonly used as protein markers of extracellular vesicles, although their role in the unexplored mechanisms of cargo selection into exosomes has not been addressed. For that purpose, we have characterized the intracellular tetraspanin-enriched microdomain (TEM) interactome by high throughput mass spectrometry, in both human lymphoblasts and their derived exosomes, revealing a clear pattern of interaction networks. Proteins interacting with TEM receptors cytoplasmic regions presented a considerable degree of overlap, although some highly specific CD81 tetraspanin ligands, such as Rac GTPase, were detected. Quantitative proteomics showed that TEM ligands account for a great proportion of the exosome proteome and that a selective repertoire of CD81-associated molecules, including Rac, is not correctly routed to exosomes in cells from CD81-deficient animals. Our data provide evidence that insertion into TEM may be necessary for protein inclusion into the exosome structure. PMID:23463506

  11. Microdomains bounded by endoplasmic reticulum segregate cell cycle calcium transients in syncytial Drosophila embryos

    PubMed Central

    Parry, Huw; McDougall, Alex; Whitaker, Michael

    2005-01-01

    Cell cycle calcium signals are generated by the inositol trisphosphate (InsP3)–mediated release of calcium from internal stores (Ciapa, B., D. Pesando, M. Wilding, and M. Whitaker. 1994. Nature. 368:875–878; Groigno, L., and M. Whitaker. 1998. Cell. 92:193–204). The major internal calcium store is the endoplasmic reticulum (ER); thus, the spatial organization of the ER during mitosis may be important in shaping and defining calcium signals. In early Drosophila melanogaster embryos, ER surrounds the nucleus and mitotic spindle during mitosis, offering an opportunity to determine whether perinuclear localization of ER conditions calcium signaling during mitosis. We establish that the nuclear divisions in syncytial Drosophila embryos are accompanied by both cortical and nuclear localized calcium transients. Constructs that chelate InsP3 also prevent nuclear division. An analysis of nuclear calcium concentrations demonstrates that they are differentially regulated. These observations demonstrate that mitotic calcium signals in Drosophila embryos are confined to mitotic microdomains and offer an explanation for the apparent absence of detectable global calcium signals during mitosis in some cell types. PMID:16216922

  12. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  13. Photophysical and electron-transfer properties of pseudoisocyanine in the hydrophobic microdomain of an aqueous polyelectrolyte

    SciTech Connect

    Jones, G. II; Oh, C. )

    1994-03-03

    The binding of pseudoisocyanine (PIC[sup +]) to the polyelectrolyte poly(methacrylic acid) (PMAA) has profound effects on the photophysical and photochemical properties of this prototypical cyanine dye. The hydrophobic dye was bound in the microdomain of the compact conformation of the polymer in its (uncharged, [open quotes]hypercoiled[close quotes]) acid form at pH < 4.0 in water. Under these conditions, the fluorescence quantum yield for PIC[sup +] was increased 600-fold and its lifetime is extended to 2.7 ns. The dye triplet state observed by flash photolysis provided a very long-lived phototransient ([lambda][sub max] = 640 nm, 50-100-[mu]s decay time). Electron-transfer quenching was investigated using the oxidant tetranitromethane (TNM) which provided the semioxidized dye radical intermediate (440-nm transient) on cobinding within PMAA hypercoils. The dye was also bound to a covalently modified form of PMAA in which polymer chains were end-labeled with 9-methylanthracene moieties. Electron transfer between anthracene chromophores and PIC[sup +] within the polymer domain was observed. 71 refs., 14 figs., 2 tabs.

  14. Plasma membrane microdomains regulate TACE-dependent TNFR1 shedding in human endothelial cells

    PubMed Central

    D’Alessio, Alessio; Esposito, Bianca; Giampietri, Claudia; Ziparo, Elio; Pober, Jordan S; Filippini, Antonio

    2012-01-01

    Abstract Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine. PMID:21645239

  15. Interplay of channels, pumps and organelle location in calcium microdomain formation

    NASA Astrophysics Data System (ADS)

    Peglow, Martin; Niemeyer, Barbara A.; Hoth, Markus; Rieger, Heiko

    2013-05-01

    To analyze the influence of Ca2+ microdomains on the global cytosolic Ca2+ concentration, we consider the polarization and activation of T-cells after the formation of an immunological synapse as a model system. For T-cell proliferation and activation, a high and robust Ca2+ signal lasting from minutes up to hours is needed. This raises the intriguing question of how T-cells overcome all those mechanisms which normally remove an increased Ca2+ level as fast as possible from the cytosol. With the help of theoretical models we predict that, after the formation of a local Ca2+ influx pathway via STIM1 and Orai1, mitochondria relocation toward and accumulation of plasma membrane Ca2+ ATPase and sarcoplasmic/ endoplasmic reticulum calcium ATPase pumps at the immunological synapse are sufficient to achieve a long-lasting increased global Ca2+ concentration. In addition, we also uncover new mechanisms to generate Ca2+ oscillations, which are important for efficient T-cell activation. Experimental tests and the implications of our predictions are discussed.

  16. Astrocyte calcium microdomains are inhibited by bafilomycin A1 and cannot be replicated by low-level Schaffer collateral stimulation in situ.

    PubMed

    Sun, Min-Yu; Devaraju, Prakash; Xie, Alison Xiaoqiao; Holman, Isabelle; Samones, Emmelyn; Murphy, Thomas R; Fiacco, Todd A

    2014-01-01

    Astrocyte Gq GPCR and IP3 receptor-dependent Ca(2+) elevations occur spontaneously in situ and in vivo. These events vary considerably in size, often remaining confined to small territories of astrocyte processes called "microdomains" and sometimes propagating over longer distances that can include the soma. It has remained unclear whether these events are driven by constitutive (basal) GPCR signaling activity, neuronal action potential-dependent or quantal vesicular release, or some combination of these mechanisms. Here, we applied manipulations to increase or inhibit neuronal vesicular neurotransmitter release together with low-level stimulation of Schaffer collaterals in acute mouse hippocampal slices in an effort to determine the mechanisms underlying spontaneous astrocyte Ca(2+) events. We found no significant change in spontaneous microdomain astrocyte Ca(2+) elevations when neuronal action potentials were significantly enhanced or blocked. The astrocyte Ca(2+) activity was also not affected by inhibitors of group I mGluRs. However, blockade of miniature neurotransmitter release using Bafilomycin A1 significantly reduced the frequency of microdomain astrocyte Ca(2+) elevations. We then tested whether astrocyte Ca(2+) microdomains can be evoked by low intensity SC stimulation. Importantly, microdomains could not be reproduced even using single, low intensity pulses to the SCs at a minimum distance from the astrocyte. Evoked astrocyte Ca(2+) responses most often included the cell soma, were reduced by group I mGluR antagonists, and were larger in size compared to spontaneous Ca(2+) microdomains. Overall, our findings suggest that spontaneous microdomain astrocyte Ca(2+) elevations are not driven by neuronal action potentials but require quantal release of neurotransmitter which cannot be replicated by stimulation of Schaffer collaterals. PMID:24262208

  17. Role of Membrane Microdomains in Compartmentation of cAMP Signaling

    PubMed Central

    Agarwal, Shailesh R.; Yang, Pei-Chi; Rice, Monica; Singer, Cherie A.; Nikolaev, Viacheslav O.; Lohse, Martin J.; Clancy, Colleen E.; Harvey, Robert D.

    2014-01-01

    Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane. PMID:24752595

  18. Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction.

    PubMed

    Curtis, Matthew W; Budyn, Elisa; Desai, Tejal A; Samarel, Allen M; Russell, Brenda

    2013-01-01

    Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100-μm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions. PMID:22407215

  19. Endothelial SK3 channel-associated Ca2+ microdomains modulate blood pressure.

    PubMed

    Yap, Fui C; Weber, David S; Taylor, Mark S; Townsley, Mary I; Comer, Brian S; Maylie, James; Adelman, John P; Lin, Mike T

    2016-05-01

    Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca(2+)-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3(-/-)) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca(2+) dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3(-/-) small mesenteric arteries. Immunostaining results from SK3(-/-) vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3(-/-) endothelial cells. Moreover, Ca(2+) imaging studies in SK3(-/-) endothelium showed increased Ca(2+) transients with reduced amplitude and duration under basal conditions. Importantly, SK3(-/-) endothelium lacked a distinct type of Ca(2+) dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3(-/-) mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca(2+) dynamics, vascular tone, and blood pressure. PMID:26945080

  20. Microdomain heterogeneity in 3D affects the mechanics of neonatal cardiac myocyte contraction

    PubMed Central

    Curtis, Matthew W.; Budyn, Elisa; Desai, Tejal A.; Samarel, Allen M.

    2012-01-01

    Cardiac muscle cells are known to adapt to their physical surroundings, optimizing intracellular organization and contractile function for a given culture environment. A previously developed in vitro model system has shown that the inclusion of discrete microscale domains (or microrods) in three dimensions (3D) can alter long-term growth responses of neonatal ventricular myocytes. The aim of this work was to understand how cellular contact with such a domain affects various mechanical changes involved in cardiac muscle cell remodeling. Myocytes were maintained in 3D gels over 5 days in the presence or absence of 100 – μm-long microrods, and the effect of this local heterogeneity on cell behavior was analyzed via several imaging techniques. Microrod abutment resulted in approximately twofold increases in the maximum displacement of spontaneously beating myocytes, as based on confocal microscopy scans of the gel xy-plane or the myocyte long axis. In addition, microrods caused significant increases in the proportion of aligned myofibrils (≤20° deviation from long axis) in fixed myocytes. Microrod-related differences in axial contraction could be abrogated by long-term interruption of certain signals of the RhoA-/Rho-associated kinase (ROCK) or protein kinase C (PKC) pathway. Furthermore, microrod-induced increases in myocyte size and protein content were prevented by ROCK inhibition. In all, the data suggest that microdomain heterogeneity in 3D appears to promote the development of axially aligned contractile machinery in muscle cells, an observation that may have relevance to a number of cardiac tissue engineering interventions. PMID:22407215

  1. Crystallization around solid-like nanosized docks can explain the specificity, diversity, and stability of membrane microdomains

    PubMed Central

    de Almeida, Rodrigo F. M.; Joly, Etienne

    2014-01-01

    To date, it is widely accepted that microdomains do form in the biological membranes of all eukaryotic cells, and quite possibly also in prokaryotes. Those sub-micrometric domains play crucial roles in signaling, in intracellular transport, and even in inter-cellular communications. Despite their ubiquitous distribution, and the broad and lasting interest invested in those microdomains, their actual nature and composition, and even the physical rules that regiment their assembly still remain elusive and hotly debated. One of the most often considered models is the raft hypothesis, i.e., the partition of lipids between liquid disordered and ordered phases (Ld and Lo, respectively), the latter being enriched in sphingolipids and cholesterol. Although it is experimentally possible to obtain the formation of microdomains in synthetic membranes through Ld/Lo phase separation, there is an ever increasing amount of evidence, obtained with a wide array of experimental approaches, that a partition between domains in Ld and Lo phases cannot account for many of the observations collected in real cells. In particular, it is now commonly perceived that the plasma membrane of cells is mostly in Lo phase and recent data support the existence of gel or solid ordered domains in a whole variety of live cells under physiological conditions. Here, we present a model whereby seeds comprised of oligomerised proteins and/or lipids would serve as crystal nucleation centers for the formation of diverse gel/crystalline nanodomains. This could confer the selectivity necessary for the formation of multiple types of membrane domains, as well as the stability required to match the time frames of cellular events, such as intra- or inter-cellular transport or assembly of signaling platforms. Testing of this model will, however, require the development of new methods allowing the clear-cut discrimination between Lo and solid nanoscopic phases in live cells. PMID:24634670

  2. Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains[S

    PubMed Central

    Betz, Josefine; Bielaszewska, Martina; Thies, Andrea; Humpf, Hans-Ulrich; Dreisewerd, Klaus; Karch, Helge; Kim, Kwang S.; Friedrich, Alexander W.; Müthing, Johannes

    2011-01-01

    Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types. PMID:21252262

  3. Insulin receptors and downstream substrates associate with membrane microdomains after treatment with insulin or chromium(III) picolinate.

    PubMed

    Al-Qatati, Abeer; Winter, Peter W; Wolf-Ringwall, Amber L; Chatterjee, Pabitra B; Van Orden, Alan K; Crans, Debbie C; Roess, Deborah A; Barisas, B George

    2012-04-01

    We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)(3)). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100 nm-scale membrane compartments after treatment with either 200 nM insulin or 10 μM Cr(pic)(3). These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)(3) but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)(3) did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)(3) likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)(3) involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)(3) are due to changes in membrane lipid order rather than to direct interactions with IR. PMID:22101510

  4. The HIV coat protein gp120 promotes forward trafficking and surface clustering of NMDA receptors in membrane microdomains

    PubMed Central

    Xu, Hangxiu; Bae, Mihyun; Tovar-y-Romo, Luis B.; Patel, Neha; Bandaru, Veera Venkata Ratnam; Pomerantz, Daniel; Steiner, Joseph; Haughey, Norman J.

    2011-01-01

    Infection by the Human immunodeficiency virus (HIV) can result in debilitating neurological syndromes collectively known as HIV associated neurocognitive disorders (HAND). While the HIV coat protein gp120 has been identified as a potent neurotoxin that enhances NMDA receptor function, the exact mechanisms for effect are not known. Here we provide evidence that gp120 activates two separate signaling pathways that converge to enhance NMDA-evoked calcium flux by clustering NMDA receptors in modified membrane microdomains. HIV gp120 enlarged, and stabilized the structure of lipid rafts on neuronal dendrites by mechanisms that involved a redox-regulated translocation of a sphingomyelin hydrolase (neutral sphingomyelinase-2; nSMase2) to the plasma membrane. A concurrent pathway was activated that enhanced the forward traffic of NMDA receptors by promoting a PKA-dependent phopshorylation of the NR1 C-terminal serine 897 (that masks an ER retention signal), followed by a PKC-dependent phosphorylation of serine 896 (important for surface expression). NMDA receptors were preferentially targeted to synapses, and clustered in modified membrane microdomains. In these conditions, NMDA receptors were unable to laterally disperse, and did not internalize, even in response to strong agonist induction. Focal NMDA-evoked calcium bursts were enhanced three-fold in these regions. Inhibiting membrane modification or NR1 phosphorylation prevented gp120 from enhancing the surface localization and clustering of NMDA receptors, while disrupting the structure of membrane microdomains restored the ability of NMDA receptors to disperse and internalize following gp120. These findings demonstrate that gp120 contributes to synaptic dysfunction in the setting of HIV-infection by interfering with the traffic of NMDA receptors. PMID:22114277

  5. Simulation strategies for calcium microdomains and calcium-regulated calcium channels.

    PubMed

    von Wegner, Frederic; Wieder, Nicolas; Fink, Rainer H A

    2012-01-01

    In this article, we present an overview of simulation strategies in the context of subcellular domains where calcium-dependent signaling plays an important role. The presentation follows the spatial and temporal scales involved and represented by each algorithm. As an exemplary cell type, we will mainly cite work done on striated muscle cells, i.e. skeletal and cardiac muscle. For these cells, a wealth of ultrastructural, biophysical and electrophysiological data is at hand. Moreover, these cells also express ubiquitous signaling pathways as they are found in many other cell types and thus, the generalization of the methods and results presented here is straightforward.The models considered comprise the basic calcium signaling machinery as found in most excitable cell types including Ca(2+) ions, diffusible and stationary buffer systems, and calcium regulated calcium release channels. Simulation strategies can be differentiated in stochastic and deterministic algorithms. Historically, deterministic approaches based on the macroscopic reaction rate equations were the first models considered. As experimental methods elucidated highly localized Ca(2+) signaling events occurring in femtoliter volumes, stochastic methods were increasingly considered. However, detailed simulations of single molecule trajectories are rarely performed as the computational cost implied is too large. On the mesoscopic level, Gillespie's algorithm is extensively used in the systems biology community and with increasing frequency also in models of microdomain calcium signaling. To increase computational speed, fast approximations were derived from Gillespie's exact algorithm, most notably the chemical Langevin equation and the τ-leap algorithm. Finally, in order to integrate deterministic and stochastic effects in multiscale simulations, hybrid algorithms are increasingly used. These include stochastic models of ion channels combined with deterministic descriptions of the calcium buffering

  6. Fractional crystallization and homogeneous nucleation of confined PEG microdomains in PBS-PEG multiblock copolymers.

    PubMed

    Huang, Cai-Li; Jiao, Ling; Zeng, Jian-Bing; Zhang, Jing-Jing; Yang, Ke-Ke; Wang, Yu-Zhong

    2013-09-12

    Fractional crystallization, homogeneous nucleation of poly(ethylene glycol) (PEG) segment, and self-nucleation behavior of PEG segment within miscible double crystalline poly(butylene succinate)-poly(ethylene glycol) (PBSEG) multiblock copolymers with different composition and segment chain length were studied by differential scanning calorimetry (DSC). Surface morphology of PBSEG10K with different PEG content was investigated by atomic force microscope (AFM). Different from di- or triblock copolymers, the microstructure and confinement of PEG dispersed phase in PBS matrix phase highly depends on chain length and sequence as well as segment content. The transition point of the PEG segment content from heterogeneous to homogeneous nucleation mechanism decreased from 50 to 39 wt % with PEG segment chain length increasing from 1000 to 2000 g/mol. When PEG segment chain length increased further to 6000 and 10000 g/mol, homogeneous nucleation phenomenon took place at much lower PEG content and fractional crystallization was observed at 29 and 24 wt %, respectively. Homogeneous nucleation mechanism of PBSEG(1K-36), PBSEG(2K-26), PBSEG(6K-19), and PBSEG(10K-12) was evidenced by the large supercoolings needed for crystallization, as well as first-order crystallization kinetics obtained. Self-nucleation behaviors of PEG segment still rely on the composition of PBSEGs. In the case of heterogeneous nucleation crystallization, self-nucleation behaviors of PEG segment showed standard self-nucleation behavior with classical three self-nucleation domains. When the crystallizable chains were confined into isolated microdomains, however, self-nucleation domain (domain II) disappeared. The absence of III(A) was observed in PBSEG(2K-39), while PBSEG(6K-29) had both III(A) and III(SA). Furthermore, AFM morphology studies still indicated the confined degree of PEG segment by previous PBS crystals was profoundly influenced by segment fraction. The confinement of the PEG segment by

  7. Rafts and the battleships of defense: the multifaceted microdomains for positive and negative signals in immune cells.

    PubMed

    Szöor, Arpád; Szöllosi, János; Vereb, György

    2010-05-01

    Recognition of the heterogeneity of the cell membrane was one of the most important scientific achievements in the last decades. Since coining the term "lipid rafts", continuous development of advanced microscopic and spectroscopic techniques has vastly expanded our view on these cell membrane microdomains that appear to have almost as many faces as researchers that look at them; they are variable in stability, size and composition that can change in a highly dynamic manner both by recruiting and expelling components as well as by coalescing and breaking up into smaller units. They have, however, one common feature: all eukaryotic cells present some variation of lipid rafts. Cells of the immune system are not exception to this, regardless of their lymphoid or myeloid origin their membranes show a domain structure and these domains serve to condense or reject particular transmembrane, GPI-linked and intracellularly membrane-anchored proteins as function requires. Here we provide a concise overview about the various weapons and shields that immune cells concentrate into their rafts, which have come into sight during the past years. The positive and negative regulatory roles of these microdomains are essential both in the functions of innate immunity and processes concatenated in the adaptive immune response. PMID:20026358

  8. Acrosome reaction-related steroidal saponin, Co-ARIS, from the starfish induces structural changes in microdomains.

    PubMed

    Naruse, Masahiro; Suetomo, Hiroyuki; Matsubara, Teruhiko; Sato, Toshinori; Yanagawa, Hiroshi; Hoshi, Motonori; Matsumoto, Midori

    2010-11-01

    Cofactor for acrosome reaction-inducing substance (Co-ARIS) is a steroidal saponin from the starfish Asterias amurensis. Saponins exist in many plants and few animals as self-defensive chemicals, but Co-ARIS has been identified as a cofactor for inducing the acrosome reaction (AR). In A. amurensis, the AR is induced by the cooperative action of egg coat components (ARIS, Co-ARIS, and asterosap); however, the mechanism of action of Co-ARIS is obscure. In this study we elucidated the membrane dynamics involved in the action of Co-ARIS. We found that cholesterol specifically inhibited the Co-ARIS activity for AR induction and detected the binding of labeled compounds with sperm using radioisotope-labeled Co-ARIS. Co-ARIS treatment did not reduce the content of sperm sterols, however, the condition was changed and localization of GM1 ganglioside on the periacrosomal region disappeared. We then developed a caveola-breaking assay, a novel method to detect the effect of chemicals on microdomains of culture cell, and confirmed the disturbance of somatic cell caveolae in the presence of Co-ARIS. Finally, by atomic force microscopy observations and surface plasmon resonance measurements using an artificial membrane, we revealed that Co-ARIS colocalized with GM1 clusters on the microdomains. Through this study, we revealed a capacitation-like event for AR in starfish sperm. PMID:20816679

  9. Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network

    PubMed Central

    Mital, Jeffrey; Miller, Natalie J.; Fischer, Elizabeth R.; Hackstadt, Ted

    2010-01-01

    Summary Chlamydiae are gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule organizing center (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability. PMID:20331642

  10. Multimerization of Human Immunodeficiency Virus Type 1 Gag Promotes Its Localization to Barges, Raft-Like Membrane Microdomains

    PubMed Central

    Lindwasser, O. Wolf; Resh, Marilyn D.

    2001-01-01

    The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed “barges.” Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process. PMID:11483736

  11. Streptococcus suis Capsular Polysaccharide Inhibits Phagocytosis through Destabilization of Lipid Microdomains and Prevents Lactosylceramide-Dependent Recognition

    PubMed Central

    Houde, Mathieu; Gottschalk, Marcelo; Gagnon, Fleur; Van Calsteren, Marie-Rose

    2012-01-01

    Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes. PMID:22124659

  12. Biochemical isolation of a membrane microdomain from resting platelets highly enriched in the plasma membrane glycoprotein CD36.

    PubMed Central

    Dorahy, D J; Lincz, L F; Meldrum, C J; Burns, G F

    1996-01-01

    Here we describe the isolation and characterization of a Triton X-100-insoluble fraction isolated from lysates of platelets by flotation in sucrose gradients. Transmission electron microscopy of the insoluble material revealed a heterogeneous population of vesicles ranging in size from 20 to 1000 nm, and Western blot analyses of platelet lysates for the caveolae structural coat protein, caveolin/VIP21, were negative. Biochemical characterization of the Triton X-100-insoluble fraction showed it to be cholesterol-rich, greatly and specifically enriched in the plasma membrane glycoprotein CD36, and also to contain Src and the Src-related kinase, Lyn. CD36 within this fraction is shown to be palmitoylated, but the fraction itself is not generally enriched in palmitoylated platelet proteins. These results suggest that this fraction represents caveolin-negative, CD36-rich microdomains in the resting platelet membrane. CD36 can form associations with certain Src-related kinases and can signal to activate platelets. These results suggest the possibility that such microdomains are implicated in platelet activation. PMID:8870650

  13. Single-Molecule Microscopy Reveals Plasma Membrane Microdomains Created by Protein-Protein Networks that Exclude or Trap Signaling Molecules in T Cells

    PubMed Central

    Douglass, Adam D.; Vale, Ronald D.

    2010-01-01

    Summary Membrane subdomains have been implicated in T cell signaling, although their properties and mechanisms of formation remain controversial. Here, we have used single-molecule and scanning confocal imaging to characterize the behavior of GFP-tagged signaling proteins in Jurkat T cells. We show that the coreceptor CD2, the adaptor protein LAT, and tyrosine kinase Lck cocluster in discrete microdomains in the plasma membrane of signaling T cells. These microdomains require protein-protein interactions mediated through phosphorylation of LAT and are not maintained by interactions with actin or lipid rafts. Using a two color imaging approach that allows tracking of single molecules relative to the CD2/LAT/Lck clusters, we demonstrate that these microdomains exclude and limit the free diffusion of molecules in the membrane but also can trap and immobilize specific proteins. Our data suggest that diffusional trapping through protein-protein interactions creates microdomains that concentrate or exclude cell surface proteins to facilitate T cell signaling. PMID:15960980

  14. The detection limit of a Gd3+-based T1 agent is substantially reduced when targeted to a protein microdomain

    PubMed Central

    Hanaoka, Kenjiro; Lubag, Angelo Josue M.; Castillo-Muzquiz, Aminta; Kodadek, Thomas; Sherry, A. Dean

    2008-01-01

    Simple low MW chelates of Gd3+ such as those currently used in clinical MR imaging are considered too insensitive for most molecular imaging applications. Here, we evaluated the detection limit of a molecularly targeted, low MW Gd3+-based, T1 agent in a model where the receptor concentration was precisely known. The data demonstrate that receptors clustered together to form a microdomain of high local concentration can be imaged successfully even when the bulk concentration of the receptor is quite low. A GdDO3A-peptide identified by phage display to target the anti-FLAG antibody was synthesized, purified and characterized. T1 weighted MR images were compared with the agent bound to antibody in bulk solution and with the agent bound to the antibody localized on agarose beads. Fluorescence competition binding assays show that the agent has a high binding affinity (KD = 150 nM) for the antibody while the fully bound relaxivity of the GdDO3A-peptide:anti-FLAG antibody in solution was a relatively modest 17 mM−1s−1. The agent:antibody complex was MR silent at concentrations below ~9 µM but was detectable down to 4 µM bulk concentrations when presented to antibody clustered together on the surface of agarose beads. These results provided an estimate of the detection limits for other T1-based agents with higher fully bound relaxivities or multimeric structures bound to clustered receptor molecules. The results demonstrate that the sensitivity of molecularly-targeted contrast agents depends on the local microdomain concentration of the target protein and the molecular relaxivity of the bound complex. A model is presented which predicts that for a molecularly targeted agent consisting of a single Gd3+ complex with bound relaxivity of 100 mM−1s−1 or, more reasonably, four tethered Gd3+ complexes each having a bound relaxivity of 25 mM−1s−1, the detection limit of a protein microdomain is ~690 nM at 9.4T. These experimental and extrapolated detection limits are

  15. Signal Transduction of Fertilization in Frog Eggs and Anti-Apoptotic Mechanism in Human Cancer Cells: Common and Specific Functions of Membrane Microdomains

    PubMed Central

    Sato, Ken-Ichi

    2008-01-01

    Membrane microdomains or lipid/membrane rafts are distinct areas on the plasma membranes, where a specific subset of lipids (e.g. cholesterol, sphingolipids) and proteins (e.g. glycosylphosphatidylinositol-anchored proteins, growth factor receptor/kinases) are getting together and functioning for several aspects of cellular functions. Our recent investigation has revealed that fertilization of African clawed frog, Xenopus laevis, requires cholesterol-dependent nature of egg membrane microdomains. Moreover, fertilization of Xenopus eggs involves proteolytic cleavage of the extracellular part and subsequent phosphorylation of a cytoplasmic tyrosine residue of uroplakin III, an egg membrane microdomain-associated protein. Protease activity toward uroplakin III seems to be derived from fertilizing sperm, while phosphorylation of uroplakin III seems to be catalyzed by the egg tyrosine kinase Src, whose activation is required for cytoplasmic rearrangement of fertilized eggs; so-called ‘egg activation’. Therefore, it is assumed that uroplakin III serves an integral part of signal transduction in fertilization of Xenopus. Our more recent study on human cancer cells has revealed that a similar but distinct scheme of signal transduction operates in anti-apoptotic growth of cells. Namely, in human bladder carcinoma cells, cooperation of uroplakin III and Src, both of which localize to the membrane microdomains, allows cells to escape from apoptotic cell death and proliferate under culture conditions deprived of serum. In this review, I briefly introduce about biology of fertilization and cancer, and then present and discuss our experimental data on general importance and specific features of membrane microdomains in Xenopus fertilization and anti-apoptosis in human bladder carcinoma cells. PMID:18949075

  16. Heterogeneous distribution of exocytotic microdomains in adrenal chromaffin cells resolved by high-density diamond ultra-microelectrode arrays

    PubMed Central

    Gosso, Sara; Turturici, Marco; Franchino, Claudio; Colombo, Elisabetta; Pasquarelli, Alberto; Carbone, Emilio; Carabelli, Valentina

    2014-01-01

    Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose–response calibrations, set the lowest detectable concentration of adrenaline to ∼5 μm. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12–27 μm2. In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The ‘non-active’ (‘silent’) zones covered 24% of the total and persisted for 6–8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution. PMID:24879870

  17. The membrane-associated form of α(s1)-casein interacts with cholesterol-rich detergent-resistant microdomains.

    PubMed

    Le Parc, Annabelle; Honvo Houéto, Edith; Pigat, Natascha; Chat, Sophie; Leonil, Joëlle; Chanat, Eric

    2014-01-01

    Caseins, the main milk proteins, interact with colloidal calcium phosphate to form the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. However, its detailed molecular organization and the cellular mechanisms involved in its biogenesis have been only partially established. There is a growing body of evidence to support the concept that α(s1)-casein takes center stage in casein micelle building and transport in the secretory pathway of mammary epithelial cells. Here we have investigated the membrane-associated form of α(s1)-casein in rat mammary epithelial cells. Using metabolic labelling we show that α(s1)-casein becomes associated with membranes at the level of the endoplasmic reticulum, with no subsequent increase at the level of the Golgi apparatus. From morphological and biochemical data, it appears that caseins are in a tight relationship with membranes throughout the secretory pathway. On the other hand, we have observed that the membrane-associated form of α(s1)-casein co-purified with detergent-resistant membranes. It was poorly solubilised by Tween 20, partially insoluble in Lubrol WX, and substantially insoluble in Triton X-100. Finally, we found that cholesterol depletion results in the release of the membrane-associated form of α(s1)-casein. These experiments reveal that the insolubility of α(s1)-casein reflects its partial association with a cholesterol-rich detergent-resistant microdomain. We propose that the membrane-associated form of α(s1)-casein interacts with the lipid microdomain, or lipid raft, that forms within the membranes of the endoplasmic reticulum, for efficient forward transport and sorting in the secretory pathway of mammary epithelial cells. PMID:25549363

  18. Heterogeneous distribution of exocytotic microdomains in adrenal chromaffin cells resolved by high-density diamond ultra-microelectrode arrays.

    PubMed

    Gosso, Sara; Turturici, Marco; Franchino, Claudio; Colombo, Elisabetta; Pasquarelli, Alberto; Carbone, Emilio; Carabelli, Valentina

    2014-08-01

    Here we describe the ability of a high-density diamond microelectrode array targeted to resolve multi-site detection of fast exocytotic events from single cells. The array consists of nine boron-doped nanocrystalline diamond ultra-microelectrodes (9-Ch NCD-UMEA) radially distributed within a circular area of the dimensions of a single cell. The device can be operated in voltammetric or chronoamperometric configuration. Sensitivity to catecholamines, tested by dose-response calibrations, set the lowest detectable concentration of adrenaline to ∼5 μm. Catecholamine release from bovine or mouse chromaffin cells could be triggered by electrical stimulation or external KCl-enriched solutions. Spikes detected from the cell apex using carbon fibre microelectrodes showed an excellent correspondence with events measured at the bottom of the cell by the 9-Ch NCD-UMEA, confirming the ability of the array to resolve single quantal secretory events. Subcellular localization of exocytosis was provided by assigning each quantal event to one of the nine channels based on its location. The resulting mapping highlights the heterogeneous distribution of secretory activity in cell microdomains of 12-27 μm2. In bovine chromaffin cells, secretion was highly heterogeneous with zones of high and medium activity in 54% of the cell surface and zones of low or no activity in the remainder. The 'non-active' ('silent') zones covered 24% of the total and persisted for 6-8 min, indicating stable location. The 9-Ch NCD-UMEA therefore appears suitable for investigating the microdomain organization of neurosecretion with high spatial resolution. PMID:24879870

  19. Cholesterol and Sphingomyelin-Containing Model Condensed Lipid Monolayers: Heterogeneities Involving Ordered Microdomains Assessed by Two Cholesterol Derivatives.

    PubMed

    Lecompte, Marie-France; Gaibelet, Gérald; Lebrun, Chantal; Tercé, François; Collet, Xavier; Orlowski, Stéphane

    2015-11-01

    Lipid monolayers are often considered as model membranes, but they are also the physiologic lipid part of the peripheral envelope of lipoproteins and cytosolic lipid bodies. However, their structural organization is still rather elusive, in particular when both cholesterol and sphingomyelin are present. To investigate such structural organization of hemimembranes, we measured, using alternative current voltammetry, the differential capacitance of condensed phosphatidylcholine-based monolayers as a function of applied potential, which is sensitive to their lipid composition and molecular arrangement. Especially, monolayers containing both sphingomyelin and cholesterol, at 15% w/w, presented specific characteristics of the differential capacitance versus potential curves recorded, which was indicative of specific interactions between these two lipid components. We then compared the behavior of two cholesterol derivatives (at 15% w/w), 21-methylpyrenyl-cholesterol (Pyr-met-Chol) and 22-nitrobenzoxadiazole-cholesterol (NBD-Chol), with that of cholesterol when present in model monolayers. Indeed, these two probes were chosen because of previous findings reporting opposite behaviors within bilayer membranes regarding their interaction with ordered lipids, with only Pyr-met-Chol mimicking cholesterol well. Remarkably, in monolayers containing sphingomyelin or not, Pyr-met-Chol and NBD-Chol presented contrasting behaviors, and Pyr-met-Chol mimicked cholesterol only in the presence of sphingomyelin. These two observations (i.e., optimal amounts of sphingomyelin and cholesterol, and the ability to discriminate between Pyr-met-Chol and NBD-Chol) can be interpreted by the existence of heterogeneities including ordered patches in sphingomyelin- and cholesterol-containing monolayers. Since such monolayer lipid arrangement shares some properties with the raft-type lipid microdomains well-described in sphingomyelin- and cholesterol-containing bilayer membranes, our data thus

  20. Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

    PubMed Central

    Wieder, Nicolas; Fink, Rainer; von Wegner, Frederic

    2015-01-01

    In this study, we numerically analyzed the nonlinear Ca2+-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP3R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca2+ buffering, and Ca2+ diffusion. The IP3R is a ubiquitous intracellular Ca2+ release channel that plays an important role in the formation of complex spatiotemporal Ca2+ signals such as waves and oscillations. Dynamic subfemtoliter Ca2+ microdomains reveal low copy numbers of Ca2+ ions, buffer molecules, and IP3Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca2+ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca2+ noise and found a distinct relation between the autocorrelation time τac, the mean channel open and close times, and the resulting IP3R open probability PO. We observed an increased PO for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca2+] = 25 μM, τac = 0.082 ms, the IP3R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca2+ binding site of IP3R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca2+ noise autocorrelation times decrease the probability of Ca2+ association and consequently increase IP3R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP3R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior

  1. Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13.

    PubMed

    Toledo, Marcos S; Suzuki, Erika; Handa, Kazuko; Hakomori, Senitiroh

    2004-08-13

    Cell growth control mechanisms were studied based on organization of components in glycosphingolipid-enriched microdomain (GEM) in WI38 cells versus their oncogenic transformant VA13 cells. Levels of fibroblast growth factor receptor (FGFR) and cSrc were 4 times and 2-3 times higher, respectively, in VA13 than in WI38 GEM, whereas the level of tetraspanin CD9/CD81 was 3-5 times higher in WI38 than in VA13 GEM. Csk, the physiological inhibitor of cSrc, was present in WI38 but not in VA13 GEM. Functional association of GEM components in control of cell growth in WI38 is indicated by several lines of evidence. (i) Confluent, growth-inhibited WI38 showed a lower degree of FGF-induced MAPK activation than actively growing cells in sparse culture. (ii) The level of inactive cSrc (with Tyr-527 phosphate) was higher in confluent cells than in actively growing cells. Both processes i and ii were inhibited by GM3 since they were enhanced by GM3 depletion with d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). (iii) The high level of inactive cSrc associated with growth-inhibited cells was caused by coexisting Csk in WI38 GEM. (iv) Interaction of GM3 with FGFR was demonstrated by binding of GM3 to FGFR in the GEM fraction, as probed with GM3-coated beads, and by confocal microscopy. In contrast to WI38, both cSrc and MAPK in VA13 were strongly activated regardless of FGF stimulation or GM3 depletion by P4. Continuous, constitutive activation of both cSrc and MAPK was due to (i) a much higher level of cSrc and FGFR in VA13 than in WI38 GEM, (ii) their close association/interaction in VA13 GEM as indicated by clear coimmunoprecipitation between cSrc and FGFR, and (iii) the absence of Csk in VA13 GEM, making GEM incapable of inhibiting cSrc activation. PMID:15143068

  2. Super-resolution imaging of ciliary microdomains in isolated olfactory sensory neurons using a custom STED microscope

    NASA Astrophysics Data System (ADS)

    Meyer, Stephanie A.; Ozbay, Baris; Restrepo, Diego; Gibson, Emily A.

    2014-03-01

    We performed super-resolution imaging of isolated olfactory sensory neurons (OSNs) using a custom-built Stimulated Emission Depletion (STED) microscope. The design for the STED microscope is based on the system developed in the laboratory of Dr. Stefan Hell1. Our system is capable of imaging with sub-diffraction limited resolution simultaneously in two color channels (at Atto 590/Atto 647N wavelengths). A single, pulsed laser source (ALP; Fianium, Inc.) generates all four laser beams, two excitation and two STED. The two STED beams are coupled into one polarization maintaining (PM) fiber and the two excitation beams into another. They are then collimated and both STED beams pass through a vortex phase plate (RPC Photonics) to allow shaping into a donut at the focus of the objective lens. The beams are then combined and sent into an inverted research microscope (IX-71; Olympus Inc.) allowing widefield epifluorescence, brightfield and DIC imaging on the same field of view as STED imaging. A fast piezo stage scans the sample during STED and confocal imaging. The fluorescent signals from the two color channels are detected with two avalanche photodiodes (APD) after appropriate spectral filtering. The resolution of the system was characterized by imaging 40 nm fluorescent beads as ~60 nm (Atto 590) and ~50 nm (Atto 647N). We performed STED imaging on immunolabeled isolated OSNs tagged at the CNGA2 and ANO2 proteins. The STED microscope allows us to resolve ciliary CNGA2 microdomains of ~54 nm that were blurred in confocal.

  3. Signaling Microdomains Regulate Inositol 1,4,5-Trisphosphate-Mediated Intracellular Calcium Transients in Cultured Neurons

    PubMed Central

    Jacob, Simon N.; Choe, Chi-Un; Uhlen, Per; DeGray, Brenda; Yeckel, Mark F.; Ehrlich, Barbara E.

    2010-01-01

    Ca2+signals in neurons use specific temporal and spatial patterns to encode unambiguous information about crucial cellular functions. To understand the molecular basis for initiation and propagation of inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular Ca2+ signals, we correlated the subcellular distribution of components of the InsP3 pathway with measurements of agonist-induced intracellular Ca2+ transients in cultured rat hippocampal neurons and pheochromocytoma cells. We found specialized domains with high levels of phosphatidylinositol-4-phosphate kinase (PIPKIγ) and chromogranin B (CGB), proteins acting synergistically to increase InsP3 pumps in the plasma membrane (PMCA) and sarco-endoplasmic reticulum receptor (InsP3R) activity and sensitivity. In contrast, Ca2+ as well as buffers that antagonize the rise in intracellular Ca2+ were distributed uniformly. By pharmacologically blocking phosphatidylinositol-4-kinase and PIPKIγ or disrupting the CGB–InsP3R interaction by transfecting an interfering polypeptide fragment, we produced major changes in the initiation site and kinetics of the Ca2+signal. This study shows that a limited number of proteins can reassemble to form unique, spatially restricted signaling domains to generate distinctive signals in different regions of the same neuron. The finding that the subcellular location of initiation sites and protein microdomains was cell type specific will help to establish differences in spatiotemporal Ca2+signaling in different types of neurons. PMID:15772345

  4. Identification, localization, and functional implications of the microdomain-forming stomatin family in the ciliated protozoan Paramecium tetraurelia.

    PubMed

    Reuter, Alexander T; Stuermer, Claudia A O; Plattner, Helmut

    2013-04-01

    The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure). PMID:23376944

  5. The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health.

    PubMed

    Ibarguren, Maitane; López, David J; Escribá, Pablo V

    2014-06-01

    This review deals with the effects of synthetic and natural fatty acids on the biophysical properties of membranes, and on their implication on cell function. Natural fatty acids are constituents of more complex lipids, like triacylglycerides or phospholipids, which are used by cells to store and obtain energy, as well as for structural purposes. Accordingly, natural and synthetic fatty acids may modify the structure of the lipid membrane, altering its microdomain organization and other physical properties, and provoking changes in cell signaling. Therefore, by modulating fatty acids it is possible to regulate the structure of the membrane, influencing the cell processes that are reliant on this structure and potentially reverting pathological cell dysfunctions that may provoke cancer, diabetes, hypertension, Alzheimer's and Parkinson's disease. The so-called Membrane Lipid Therapy offers a strategy to regulate the membrane composition through drug administration, potentially reverting pathological processes by re-adapting cell membrane structure. Certain fatty acids and their synthetic derivatives are described here that may potentially be used in such therapies, where the cell membrane itself can be considered as a target to combat disease. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. PMID:24388951

  6. Identification, Localization, and Functional Implications of the Microdomain-Forming Stomatin Family in the Ciliated Protozoan Paramecium tetraurelia

    PubMed Central

    Stuermer, Claudia A. O.; Plattner, Helmut

    2013-01-01

    The SPFH protein superfamily is assumed to occur universally in eukaryotes, but information from protozoa is scarce. In the Paramecium genome, we found only Stomatins, 20 paralogs grouped in 8 families, STO1 to STO8. According to cDNA analysis, all are expressed, and molecular modeling shows the typical SPFH domain structure for all subgroups. For further analysis we used family-specific sequences for fluorescence and immunogold labeling, gene silencing, and functional tests. With all family members tested, we found a patchy localization at/near the cell surface and on vesicles. The Sto1p and Sto4p families are also associated with the contractile vacuole complex. Sto4p also makes puncta on some food vacuoles and is abundant on vesicles recycling from the release site of spent food vacuoles to the site of nascent food vacuole formation. Silencing of the STO1 family reduces mechanosensitivity (ciliary reversal upon touching an obstacle), thus suggesting relevance for positioning of mechanosensitive channels in the plasmalemma. Silencing of STO4 members increases pulsation frequency of the contractile vacuole complex and reduces phagocytotic activity of Paramecium cells. In summary, Sto1p and Sto4p members seem to be involved in positioning specific superficial and intracellular microdomain-based membrane components whose functions may depend on mechanosensation (extracellular stimuli and internal osmotic pressure). PMID:23376944

  7. Anchorage of HIV on permissive cells leads to coaggregation of viral particles with surface nucleolin at membrane raft microdomains.

    PubMed

    Nisole, Sébastien; Krust, Bernard; Hovanessian, Ara G

    2002-06-10

    The cross-linking of HIV on permissive cells results aggregation of HIV particles with surface nucleolin, CD4, and CXCR4, but without affecting the organization of CD45. In addition, HIV particles and nucleolin coaggregate with glycolipid-enriched membrane microdomains (GEMs) containing ganglioside, and glycosylphosphatidylinositol-linked proteins CD90 and CD59, pointing out that HIV anchorage induces lateral assemblies of specific membrane components into lipid rafts in which surface nucleolin is also incorporated. Consequently, equilibrium density fractionation of extracts from infected cells revealed that HIV proteins and nucleolin copurify with Triton X-100-resistant GEM-associated proteins. After HIV entry, nucleolin is recovered also in fractions containing HIV DNA, viral matrix, and reverse transcriptase, thus suggesting that it could accompany viral entry. We show that surface nucleolin is markedly down-regulated a few hours following HIV entry into permissive cells; an effect that appears to be the consequence of its translocation into the cytoplasm. Our findings demonstrate that anchorage of HIV particles on permissive cells induces aggegation of surface nucleolin and its association with detergent-insoluble lipid raft components. Moreover, they support the suggestion that surface nucleolin and lipid rafts are implicated in early events in the HIV entry process. PMID:12027446

  8. Sphingosine Kinase 1 Localized to the Plasma Membrane Lipid Raft Microdomain Overcomes Serum Deprivation Induced Growth Inhibition

    PubMed Central

    Hengst, Jeremy A.; Francy-Guilford, Jacquelyn M.; Fox, Todd E.; Wang, Xujun; Conroy, Elizabeth J.; Yun, Jong K.

    2009-01-01

    Several studies have demonstrated that sphingosine kinase 1 (SphK1) translocates to the plasma membrane (PM) upon its activation and further suggested the plasma membrane lipid raft microdomain (PMLRM) as a target for SphK1 relocalization. To date, however, direct evidence of SphK1 localization to the PMLRM has been lacking. In this report, using multiple biochemical and subcellular fractionation techniques we demonstrate that endogenous SphK1 protein and its substrate, D-erythro sphingosine, are present within the PMLRM. Additionally, we demonstrate that the PMA stimulation of SphK1 localized to the PMLRM results in production of sphingosine-1-phosphate as well as induction of cell growth under serum-deprivation conditions. We further report that Ser225Ala and Thr54Cys mutations, reported to abrogate phosphatidylserine binding, block SphK1 targeting to the PMLRM and SphK1 induced cell growth. Together these findings provide direct evidence that the PMLRM is the major site-of-action for SphK1 to overcome serum-deprived cell growth inhibition. PMID:19782042

  9. Molecularly designed lipid microdomains for solid dispersions using a polymer/inorganic carrier matrix produced by hot-melt extrusion.

    PubMed

    Adler, Camille; Schönenberger, Monica; Teleki, Alexandra; Kuentz, Martin

    2016-02-29

    Amorphous solid dispersions have for many years been a focus in oral formulations, especially in combination with a hot-melt extrusion process. The present work targets a novel approach with a system based on a fatty acid, a polymer and an inorganic carrier. It was intended to adsorb the acidic lipid by specific molecular interactions onto the solid carrier to design disorder in the alkyl chains of the lipid. Such designed lipid microdomains (DLM) were created as a new microstructure to accommodate a compound in a solid dispersion. Vibrational spectroscopy, X-ray powder diffraction, atomic force microscopy as well as electron microscopic imaging were employed to study a system of stearic acid, hydroxypropylcellulose and aluminum magnesium silicate. β-carotene was used as a poorly water-soluble model substance that is difficult to formulate with conventional solid dispersion formulations. The results indicated that the targeted molecular excipient interactions indeed led to DLMs for specific compositions. The different methods provided complementary aspects and important insights into the created microstructure. The novel delivery system appeared to be especially promising for the formulation of oral compounds that exhibit both high crystal energy and lipophilicity. PMID:26721729

  10. Caveolae from luminal plasmalemma of rat lung endothelium: microdomains enriched in caveolin, Ca(2+)-ATPase, and inositol trisphosphate receptor.

    PubMed Central

    Schnitzer, J E; Oh, P; Jacobson, B S; Dvorak, A M

    1995-01-01

    A distinctive feature of many endothelia is an abundant population of noncoated plasmalemmal vesicles, or caveolae. Caveolae have been implicated in many important cellular processes, including transcytosis, endocytosis, potocytosis, and even signal transduction. Because caveolae have not been purified from endothelial cell surfaces, little is known directly about their structure and function in the endothelium. To delineate the transport role of these caveolae, we purified them from isolated luminal endothelial plasma membranes of rat lung. The rat lung luminal endothelial cell surfaces were isolated after coating them, in situ, with positively charged colloidal silica. The caveolae were then separated from these coated membranes and purified to yield a homogeneous population of morphologically distinct vesicles enriched in the structural protein caveolin. As with caveolae found on the endothelial cell surface in vivo, these highly purified caveolae contained the plasmalemmal Ca(2+)-ATPase and inositol 1,4,5-trisphosphate surface receptors. By contrast, other plasma membrane proteins were excluded from the caveolae, including angiotensin-converting enzyme, beta-actin, and band 4.1. The purified caveolae appeared to represent specific microdomains of the cell surface with their own unique molecular topography. Images Fig. 2 Fig. 3 Fig. 5 PMID:7878055

  11. Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives.

    PubMed

    Ibarguren, Maitane; López, David J; Encinar, José A; González-Ros, José M; Busquets, Xavier; Escribá, Pablo V

    2013-11-01

    Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses. PMID:23792066

  12. Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins

    PubMed Central

    Dempwolff, Felix; Schmidt, Felix K.; Hervás, Ana B.; Stroh, Alex; Rösch, Thomas C.; Riese, Cornelius N.; Dersch, Simon; Heimerl, Thomas; Lucena, Daniella; Hülsbusch, Nikola; Stuermer, Claudia A. O.; Takeshita, Norio; Fischer, Reinhard; Graumann, Peter L.

    2016-01-01

    Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds. PMID:27362352

  13. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

    PubMed

    Vermeer, J E M; Van Munster, E B; Vischer, N O; Gadella, T W J

    2004-05-01

    Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts. PMID:15102066

  14. Large-conductance calcium-activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains.

    PubMed

    Kaufmann, Walter A; Ferraguti, Francesco; Fukazawa, Yugo; Kasugai, Yu; Shigemoto, Ryuichi; Laake, Petter; Sexton, Joseph A; Ruth, Peter; Wietzorrek, Georg; Knaus, Hans-Günther; Storm, Johan F; Ottersen, Ole Petter

    2009-07-10

    Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells. PMID:19412945

  15. The Influence of Ca2+ Buffers on Free [Ca2+] Fluctuations and the Effective Volume of Ca2+ Microdomains

    PubMed Central

    Weinberg, Seth H.; Smith, Gregory D.

    2014-01-01

    Intracellular calcium (Ca2+) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca2+ binding proteins (Ca2+ buffers) play an important role in regulating Ca2+ concentration ([Ca2+]). Buffers typically slow [Ca2+] temporal dynamics and increase the effective volume of Ca2+ domains. Because fluctuations in [Ca2+] decrease in proportion to the square-root of a domain’s physical volume, one might conjecture that buffers decrease [Ca2+] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca2+ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca2+ influx with passive exchange of both Ca2+ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca2+ and buffer and use these stochastic differential equations to determine the magnitude of [Ca2+] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca2+ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca2+] fluctuations during periods of Ca2+ influx, whereas stationary (immobile) Ca2+ buffers do not. Also contrary to expectations, we find that in the absence of Ca2+ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca2+] fluctuations. We derive an analytical formula describing the influence of rapid Ca2+ buffers on [Ca2+] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca2+ buffers alter the dynamics of [Ca2+] fluctuations in a nonintuitive manner. The finding that Ca2+ buffers do not suppress intrinsic domain [Ca2

  16. Microdomain Formation, Oxidation, and Cation Ordering in LaCa2Fe3O8+y

    SciTech Connect

    Price, Patrick M.; Browning, Nigel D.; Butt, Darryl P.

    2015-03-23

    The compound LaCa2Fe3O8+y, also known as the Grenier phase, is known to undergo an order-disorder transformation (ODT) at high temperatures. Oxidation has been observed when the compound is cooled in air after the ODT. In this study, we have synthesized the Grenier compound in air using traditional solid state reactions and investigated the structure and composition before and after the ODT. Thermal analysis showed that the material undergoes an order-disorder transformation in both oxygen and argon atmospheres with dynamic, temperature dependent, oxidation upon cooling. Results from scanning transmission electron microscopy (STEM) suggest that the Grenier phase has preferential segregation of Ca and La on the two crystallographic A-sites before the ODT, but a random distribution above the ODT temperature. Furthermore, STEM images suggest the possibility that oxygen excess may exist in La-rich regions within microdomains rather than at microdomain boundaries.

  17. Compartmentalized Cyclic Adenosine 3′,5′-Monophosphate at the Plasma Membrane Clusters PDE3A and Cystic Fibrosis Transmembrane Conductance Regulator into Microdomains

    PubMed Central

    Penmatsa, Himabindu; Zhang, Weiqiang; Yarlagadda, Sunitha; Li, Chunying; Conoley, Veronica G.; Yue, Junming; Bahouth, Suleiman W.; Buddington, Randal K.; Zhang, Guangping; Nelson, Deborah J.; Sonecha, Monal D.; Manganiello, Vincent; Wine, Jeffrey J.

    2010-01-01

    Formation of multiple-protein macromolecular complexes at specialized subcellular microdomains increases the specificity and efficiency of signaling in cells. In this study, we demonstrate that phosphodiesterase type 3A (PDE3A) physically and functionally interacts with cystic fibrosis transmembrane conductance regulator (CFTR) channel. PDE3A inhibition generates compartmentalized cyclic adenosine 3′,5′-monophosphate (cAMP), which further clusters PDE3A and CFTR into microdomains at the plasma membrane and potentiates CFTR channel function. Actin skeleton disruption reduces PDE3A–CFTR interaction and segregates PDE3A from its interacting partners, thus compromising the integrity of the CFTR-PDE3A–containing macromolecular complex. Consequently, compartmentalized cAMP signaling is lost. PDE3A inhibition no longer activates CFTR channel function in a compartmentalized manner. The physiological relevance of PDE3A–CFTR interaction was investigated using pig trachea submucosal gland secretion model. Our data show that PDE3A inhibition augments CFTR-dependent submucosal gland secretion and actin skeleton disruption decreases secretion. PMID:20089840

  18. KSHV attachment and entry are dependent on αVβ3 integrin localized to specific cell surface microdomains and do not correlate with the presence of heparan sulfate

    PubMed Central

    Garrigues, H. Jacques; DeMaster, Laura K.; Rubinchikova, Yelena E.; Rose, Timothy M.

    2014-01-01

    Cellular receptors for KSHV attachment and entry were characterized using tyramide signal amplification (TSA)-enhanced confocal microscopy. Integrins αVβ3, αVβ5 and α3β1 were detected on essentially all the actin-based cell surface microdomains that initially bind KSHV, while the presence of CD98 and heparan sulfate (HS), the putative attachment receptor, was more variable. KSHV bound to the same cell surface microdomains with and without HS indicating that initial attachment of KSHV is not dependent on HS and that receptors other than HS can mediate attachment. A human salivary gland (HSG) epithelial line was identified, which lacks αVβ3 but expresses high levels of HS, α3β1 and other putative KSHV receptors. These cells were resistant to KSHV-binding and infection. Reconstitution of cell surface αVβ3 rendered HSG cells highly susceptible to KSHV infection, demonstrating a critical role for αVβ3 in the binding and entry of KSHV that is not shared with other proposed receptors. PMID:25063885

  19. Control points within the cell cycle

    SciTech Connect

    Van't Hof, J.

    1984-01-01

    Evidence of the temporal order of chromosomal DNA replication argues favorably for the view that the cell cycle is controlled by genes acting in sequence whose time of expression is determined by mitosis and the amount of nuclear DNA (2C vs 4C) in the cell. Gl and G2 appear to be carbohydrate dependent in that cells starved of either carbohydrate of phosphate fail to make these transitions. Cells deprived of nitrate, however, fail only at Gl to S transition indicating that the controls that operate in G1 differ from those that operate in G2. 46 references, 5 figures.

  20. Regulation of H-Ras-driven MAPK signaling, transformation and tumorigenesis, but not PI3K signaling and tumor progression, by plasma membrane microdomains.

    PubMed

    Michael, J V; Wurtzel, J G T; Goldfinger, L E

    2016-01-01

    In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and R-Ras. These paralogs have identical effector-binding regions, but variant C-terminal targeting domains (tDs) which are responsible for lateral microdomain distribution: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, and R-Ras to Lo domains (rafts). We hypothesized that PM distribution regulates Ras-effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on signal transduction, cell proliferation, transformation and tumorigenesis. R-Ras harboring the H-Ras tD (R-Ras-tH) interacted with Raf, and induced Raf and ERK phosphorylation similar to H-Ras. R-Ras-tH stimulated proliferation and transformation in vitro, and these effects were blocked by both MEK and PI3K inhibition. Conversely, the R-Ras tD suppressed H-Ras-mediated Raf activation and ERK phosphorylation, proliferation and transformation. Thus, Ras access to Raf at the PM is sufficient for MAPK activation and is a principal component of Ras mitogenesis and transformation. Fusion of the R-Ras extended N-terminal domain to H-Ras had no effect on proliferation, but inhibited transformation and tumor progression, indicating that the R-Ras N-terminus also contributes negative regulation to these Ras functions. PI3K activation was tD independent; however, H-Ras was a stronger activator of PI3K than R-Ras, with either tD. PI3K inhibition nearly ablated transformation by R-Ras-tH, H-Ras and H-Ras-tR, whereas MEK inhibition had a modest effect on Ras-tH-driven transformation but no effect on H-Ras-tR transformation. R-Ras-tH supported tumor initiation, but not tumor progression. While H-Ras-tR-induced transformation was reduced relative to H-Ras, tumor progression was robust and similar to H-Ras. H-Ras tumor growth was moderately suppressed by MEK inhibition, which had no effect on H-Ras-tR tumor growth. In contrast, PI3K inhibition

  1. Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids.

    PubMed Central

    Lehtonen, J Y; Holopainen, J M; Kinnunen, P K

    1996-01-01

    , neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes. PMID:8785334

  2. Inhibition of VEGF-dependent angiogenesis by the anti-CD82 monoclonal antibody 4F9 through regulation of lipid raft microdomains.

    PubMed

    Nomura, Sayaka; Iwata, Satoshi; Hatano, Ryo; Komiya, Eriko; Dang, Nam H; Iwao, Noriaki; Ohnuma, Kei; Morimoto, Chikao

    2016-05-20

    CD82 (also known as KAI1) belongs to the tetraspanin superfamily of type III transmembrane proteins, and is involved in regulating cell adhesion, migration and proliferation. In contrast to these well-established roles of CD82 in tumor biology, its function in endothelial cell (EC) activity and tumor angiogenesis is yet to be determined. In this study, we show that suppression of CD82 negatively regulates vascular endothelial growth factor (VEGF)-induced angiogenesis. Moreover, we demonstrate that the anti-CD82 mAb 4F9 effectively inhibits phosphorylation of VEGF receptor 2 (VEGFR2), which is the principal mediator of the VEGF-induced angiogenic signaling process in tumor angiogenesis, by regulating the organization of the lipid raft microdomain signaling platform in human EC. Our present work therefore suggests that CD82 on EC is a potential target for anti-angiogenic therapy in VEGFR2-dependent tumor angiogenesis. PMID:27103437

  3. Segregative clustering of Lo and Ld membrane microdomains induced by local pH gradients in GM1-containing giant vesicles: a lipid model for cellular polarization.

    PubMed

    Staneva, Galya; Puff, Nicolas; Seigneuret, Michel; Conjeaud, Hélène; Angelova, Miglena I

    2012-11-27

    Several cell polarization processes are coupled to local pH gradients at the membrane surface. We have investigated the involvement of a lipid-mediated effect in such coupling. The influence of lateral pH gradients along the membrane surface on lipid microdomain dynamics in giant unilamellar vesicles containing phosphatidylcholine, sphingomyelin, cholesterol, and the ganglioside GM1 was studied. Lo/Ld phase separation was generated by photosensitization. A lateral pH gradient was established along the external membrane surface by acid local microinjection. The gradient promotes the segregation of microdomains: Lo domains within an Ld phase move toward the higher pH side, whereas Ld domains within an Lo phase move toward the lower pH side. This results in a polarization of the vesicle membrane into Lo and Ld phases poles in the axis of the proton source. A secondary effect is inward tubulation in the Ld phase. None of these processes occurs without GM1 or with the analog asialo-GM1. These are therefore related to the acidic character of the GM1 headgroup. LAURDAN fluorescence experiments on large unilamellar vesicles indicated that, with GM1, an increase in lipid packing occurs with decreasing pH, attributed to the lowering of repulsion between GM1 molecules. Packing increase is much higher for Ld phase vesicles than for Lo phase vesicles. It is proposed that the driving forces for domain vectorial segregative clustering and vesicle polarization are related to such differences in packing variations with pH decrease between the Lo and Ld phases. Such pH-driven domain clustering might play a role in cellular membrane polarization processes in which local lateral pH gradients are known to be important, such as migrating cells and epithelial cells. PMID:23121205

  4. Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes

    PubMed Central

    Glukhov, Alexey V.; Balycheva, Marina; Sanchez-Alonso, Jose L.; Ilkan, Zeki; Alvarez-Laviada, Anita; Bhogal, Navneet; Diakonov, Ivan; Schobesberger, Sophie; Sikkel, Markus B.; Bhargava, Anamika; Faggian, Giuseppe; Punjabi, Prakash P.; Houser, Steven R.

    2015-01-01

    Background— Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). Methods and Results— Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈30% whole-cell ICa,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude. Conclusions— We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner. PMID:26450916

  5. Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus

    PubMed Central

    Akiyama, Hisashi; Miller, Caitlin; Patel, Hiren V.; Hatch, Steven C.; Archer, Jacob; Ramirez, Nora-Guadalupe P.

    2014-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently reported that the capture of HIV-1 by mature dendritic cells (MDCs) is mediated by an interaction between the glycosphingolipid (GSL) GM3 on virus particles and CD169/Siglec-1 on MDCs. Since HIV-1 preferentially buds from GSL-enriched lipid microdomains on the plasma membrane, we hypothesized that the virus assembly and budding site determines the ability of HIV-1 to interact with MDCs. In support of this hypothesis, mutations in the N-terminal basic domain (29/31KE) or deletion of the membrane-targeting domain of the HIV-1 matrix (MA) protein that altered the virus assembly and budding site to CD63+/Lamp-1-positive intracellular compartments resulted in lower levels of virion incorporation of GM3 and attenuation of virus capture by MDCs. Furthermore, MDC-mediated capture and transmission of MA mutant viruses to T cells were decreased, suggesting that HIV-1 acquires GSLs via budding from the plasma membrane to access the MDC-dependent trans infection pathway. Interestingly, MDC-mediated capture of Nipah and Hendra virus (recently emerged zoonotic paramyxoviruses) M (matrix) protein-derived virus-like particles that bud from GSL-enriched plasma membrane microdomains was also dependent on interactions between virion-incorporated GSLs and CD169. Moreover, capture and transfer of Nipah virus envelope glycoprotein-pseudotyped lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination. IMPORTANCE Dendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive

  6. Ethanol Enhances TGF-β Activity by Recruiting TGF-β Receptors From Intracellular Vesicles/Lipid Rafts/Caveolae to Non-Lipid Raft Microdomains.

    PubMed

    Huang, Shuan Shian; Chen, Chun-Lin; Huang, Franklin W; Johnson, Frank E; Huang, Jung San

    2016-04-01

    Regular consumption of moderate amounts of ethanol has important health benefits on atherosclerotic cardiovascular disease (ASCVD). Overindulgence can cause many diseases, particularly alcoholic liver disease (ALD). The mechanisms by which ethanol causes both beneficial and harmful effects on human health are poorly understood. Here we demonstrate that ethanol enhances TGF-β-stimulated luciferase activity with a maximum of 0.5-1% (v/v) in Mv1Lu cells stably expressing a luciferase reporter gene containing Smad2-dependent elements. In Mv1Lu cells, 0.5% ethanol increases the level of P-Smad2, a canonical TGF-β signaling sensor, by ∼2-3-fold. Ethanol (0.5%) increases cell-surface expression of the type II TGF-β receptor (TβR-II) by ∼2-3-fold from its intracellular pool, as determined by I(125) -TGF-β-cross-linking/Western blot analysis. Sucrose density gradient ultracentrifugation and indirect immunofluorescence staining analyses reveal that ethanol (0.5% and 1%) also displaces cell-surface TβR-I and TβR-II from lipid rafts/caveolae and facilitates translocation of these receptors to non-lipid raft microdomains where canonical signaling occurs. These results suggest that ethanol enhances canonical TGF-β signaling by increasing non-lipid raft microdomain localization of the TGF-β receptors. Since TGF-β plays a protective role in ASCVD but can also cause ALD, the TGF-β enhancer activity of ethanol at low and high doses appears to be responsible for both beneficial and harmful effects. Ethanol also disrupts the location of lipid raft/caveolae of other membrane proteins (e.g., neurotransmitter, growth factor/cytokine, and G protein-coupled receptors) which utilize lipid rafts/caveolae as signaling platforms. Displacement of these membrane proteins induced by ethanol may result in a variety of pathologies in nerve, heart and other tissues. J. Cell. Biochem. 117: 860-871, 2016. © 2015 Wiley Periodicals, Inc. PMID:26419316

  7. A Shotgun Proteomic Approach Reveals That Fe Deficiency Causes Marked Changes in the Protein Profiles of Plasma Membrane and Detergent-Resistant Microdomain Preparations from Beta vulgaris Roots.

    PubMed

    Gutierrez-Carbonell, Elain; Takahashi, Daisuke; Lüthje, Sabine; González-Reyes, José Antonio; Mongrand, Sébastien; Contreras-Moreira, Bruno; Abadía, Anunciación; Uemura, Matsuo; Abadía, Javier; López-Millán, Ana Flor

    2016-08-01

    In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency. PMID:27321140

  8. Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

    PubMed

    Fujimoto, Yasuyuki; Kamakura, Aya; Motohashi, Yu; Ohashi-Kobayashi, Ayako; Maeda, Masatomo

    2011-01-01

    The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-β-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol. PMID:21212514

  9. Micro-domain analysis of skin samples of moor-mummified corpses by evanescent wave infrared spectroscopy using silver halide fibers

    NASA Astrophysics Data System (ADS)

    Küpper, L.; Heise, H. M.; Bechara, F.-G.; Stücker, M.

    2001-05-01

    Infrared microscopy plays an important role in chemical micro-domain analysis of inhomogeneous materials. A simple experimental arrangement based on fiber-optics, employing bent silver halide fibers of sub-millimeter diameter cross-section, was used for infrared ATR-measurements with a minimum spot size of 20×60 μm 2. It was applied for the analysis of skin specimens and hair samples of mummified corpses, preserved under bog conditions. The desiccated dermis samples looked leather-like, but were rather brittle, so that micro-ATR measurements by a fiber probe were appropriate. Comparable ATR-results were obtained using an IR-microscope. Composition along perpendicular dermis cross-sections was evaluated by comparison with spectra from reference materials. Natural dermis samples are mainly composed of collagen, primarily of type I and III, which was still found in the mummies' skin. The surfaces of the skin samples displayed chemical changes from moor constituents, while the center of the dermis cross-section consisted of unmodified collagen. Keratin in hair samples was also well preserved apart from surface changes, which had been caused by the bog chemistry in these samples and are clearly manifested in the infrared spectra.

  10. Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

    PubMed Central

    Rouquette-Jazdanian, Alexandre K; Pelassy, Claudette; Breittmayer, Jean-Philippe; Cousin, Jean-Louis; Aussel, Claude

    2002-01-01

    Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts. PMID:11964165

  11. Reversible cross-linking, microdomain structure, and heterogeneous dynamics in thermally reversible cross-linked polyurethane as revealed by solid-state NMR.

    PubMed

    Zhang, Rongchun; Yu, Shen; Chen, Shengli; Wu, Qiang; Chen, Tiehong; Sun, Pingchuan; Li, Baohui; Ding, Datong

    2014-01-30

    Polyurethane material is widely utilized in industry and daily life due to its versatile chemistry and relatively easy handling. Here, we focused on a novel thermally reversible cross-linked polyurethane with comprehensive remarkable mechanical properties as reported in our recent work (Adv. Mater. 2013, 25, 4912). The microphase-separated structure and heterogeneous segmental dynamics were well revealed by T2 relaxometry experiments, which was also first utilized to in situ monitor the reversible cross-linking associated with Diels-Alder (DA) and retro-Diels-Alder (RDA) reactions. On the basis of T2 relaxometry results, we determined the actual temperature of the (R)DA reaction as well as the corresponding activation energies of the motion of soft segments. Besides, the roles of the temperature and cross-linker contents on the microdomain structure and dynamics are discussed in detail. It is found that the microphase separation is enhanced by the increase of temperature as well as the incorporation of cross-linkers. Also, the polyurethane samples are still thermal-stable even at a high temperature beyond the disassociation of the cross-linkages. Furthermore, Baum-Pines and three-pulse multiple-quantum NMR experiments are utilized to investigate the heterogeneous structures and dynamics of the mobile and rigid segments, respectively. Both the results obtained from the T2 relaxometry and multiple-quantum NMR experiments are in good agreement with the macroscopic mechanical properties of the polyurethane. Finally, it is also well demonstrated that proton T2 relaxometry combined with multiple-quantum NMR is a powerful method to study the heterogeneous structures and dynamics of a multiphase polymer system. PMID:24400980

  12. Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity.

    PubMed

    Rauch, Susanne; Pulkkinen, Kati; Saksela, Kalle; Fackler, Oliver T

    2008-03-01

    Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and betaPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or betaPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a betaPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or betaPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex. PMID:18094167

  13. MreB-Dependent Organization of the E. coli Cytoplasmic Membrane Controls Membrane Protein Diffusion.

    PubMed

    Oswald, Felix; Varadarajan, Aravindan; Lill, Holger; Peterman, Erwin J G; Bollen, Yves J M

    2016-03-01

    The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs. PMID:26958890

  14. Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites.

    PubMed

    Norregaard, L; Visiers, I; Loland, C J; Ballesteros, J; Weinstein, H; Gether, U

    2000-12-26

    Previously, we have identified three Zn(2+) binding residues in an endogenous Zn(2+) binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn(2+) binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn(2+)-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn(2+) potently inhibited [(3)H]dopamine uptake with an IC(50) value of 7 microM as compared to a value of 300 microM for the control (H193K). These data are consistent with the presence of an alpha-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn(2+) affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [(3)H]dopamine uptake by Zn(2+) (IC(50) = 3-32 microM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn(2+) binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure. PMID:11123909

  15. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo

    PubMed Central

    Tsui, Cynthia C.; Gabreski, Nicole A.; Hein, Sarah J.

    2015-01-01

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF–Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1–TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1–TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1TM/TM mice, GFRα1–TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1+/TM mice are viable, Gfrα1TM/TM mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1−/− mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. SIGNIFICANCE STATEMENT Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  16. Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

    PubMed

    Tsui, Cynthia C; Gabreski, Nicole A; Hein, Sarah J; Pierchala, Brian A

    2015-09-23

    Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex

  17. Very-long-chain fatty acid sphingomyelin in nuclear lipid microdomains of hepatocytes and hepatoma cells: can the exchange from C24:0 to C16:0 affect signal proteins and vitamin D receptor?

    PubMed

    Lazzarini, Andrea; Macchiarulo, Antonio; Floridi, Alessandro; Coletti, Alice; Cataldi, Samuela; Codini, Michela; Lazzarini, Remo; Bartoccini, Elisa; Cascianelli, Giacomo; Ambesi-Impiombato, Francesco Saverio; Beccari, Tommaso; Curcio, Francesco; Albi, Elisabetta

    2015-07-01

    Lipid microdomains localized in the inner nuclear membrane are considered platforms for active chromatin anchoring. Stimuli such as surgery, vitamin D, or glucocorticoid drugs influence their gene expression, DNA duplication, and RNA synthesis. In this study, we used ultrafast liquid chromatography-tandem mass spectrometry to identify sphingomyelin (SM) species coupled with immunoblot analysis to comprehensively map differences in nuclear lipid microdomains (NLMs) purified from hepatocytes and hepatoma cells. We showed that NLMs lost saturated very-long-chain fatty acid (FA; C24:0) SM in cancer cells and became enriched in long-chain FA (C16:0) SM. We also found that signaling proteins, such as STAT3, Raf1, and PKCζ, were increased and vitamin D receptor was reduced in cancer cells. Because recent researches showed a shift in sphingolipid composition from C24:0 to C16:0 in relation to cell life, we performed a comparative analysis of properties among C16:0 SM, C24:0 SM, and cholesterol. Our results led us to hypothesize that the enrichment of C16:0 SM could determine enhanced dynamic properties of NLMs in cancer cells with an increased shuttling of protein signaling molecules. PMID:26124436

  18. Altering fatty acid availability does not impair prolonged, continuous running to fatigue: evidence for carbohydrate dependence.

    PubMed

    Leckey, Jill J; Burke, Louise M; Morton, James P; Hawley, John A

    2016-01-15

    We determined the effect of suppressing lipolysis via administration of nicotinic acid (NA) on fuel substrate selection and half-marathon running capacity. In a single-blinded, Latin square design, 12 competitive runners completed four trials involving treadmill running until volitional fatigue at a pace based on 95% of personal best half-marathon time. Trials were completed in a fed or overnight fasted state: 1) carbohydrate (CHO) ingestion before (2 g CHO·kg(-1)·body mass(-1)) and during (44 g/h) [CFED]; 2) CFED plus NA ingestion [CFED-NA]; 3) fasted with placebo ingestion during [FAST]; and 4) FAST plus NA ingestion [FAST-NA]. There was no difference in running distance (CFED, 21.53 ± 1.07; CFED-NA, 21.29 ± 1.69; FAST, 20.60 ± 2.09; FAST-NA, 20.11 ± 1.71 km) or time to fatigue between the four trials. Concentrations of plasma free fatty acids (FFA) and glycerol were suppressed following NA ingestion irrespective of preexercise nutritional intake but were higher throughout exercise in FAST compared with all other trials (P < 0.05). Rates of whole-body CHO oxidation were unaffected by NA ingestion in the CFED and FAST trials, but were lower in the FAST trial compared with the CFED-NA trial (P < 0.05). CHO was the primary substrate for exercise in all conditions, contributing 83-91% to total energy expenditure with only a small contribution from fat-based fuels. Blunting the exercise-induced increase in FFA via NA ingestion did not impair intense running capacity lasting ∼85 min, nor did it alter patterns of substrate oxidation in competitive athletes. Although there was a small but obligatory use of fat-based fuels, the oxidation of CHO-based fuels predominates during half-marathon running. PMID:26586912

  19. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-01

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo. PMID:26468279

  20. Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain*

    PubMed Central

    Sammels, Eva; Devogelaere, Benoit; Mekahli, Djalila; Bultynck, Geert; Missiaen, Ludwig; Parys, Jan B.; Cai, Yiqiang; Somlo, Stefan; De Smedt, Humbert

    2010-01-01

    Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease. PMID:20375013

  1. Characterization of electron-beam recorded microdomain patterns on the nonpolar surface of LiNbO{sub 3} crystal by nondestructive methods

    SciTech Connect

    Kokhanchik, L. S.; Gainutdinov, R. V.; Volk, T. R.; Mishina, E. D.; Lavrov, S. D.

    2014-10-06

    We report on characterization of the electron-beam fabricated planar domain gratings on the nonpolar (Y-) surface of LiNbO{sub 3} crystals performed with the use of AFM and confocal second harmonic generation (SHG) microscopy. The dependence of domain formation on the irradiation conditions was investigated. The relation of domain thicknesses to the electron penetration depth is experimentally proved. In particular, the possibility of controlling the thickness of planar domains by varying acceleration electron-beam voltages is demonstrated. The observed specificity of SHG is analyzed in the framework of the Kleinman-Boyd theory [G. D. Boyd and D. A. Kleinman, J. Appl. Phys. 39, 3597 (1968)] and Uesu approach [Kaneshiro et al., J. Appl. Phys. 104, 054112 (2008); Kaneshiro et al., J. Opt. Soc. Am. B 27, 888 (2010)] extended in our case to reflection geometry. The calculations performed predict the dependence of SHG conversion efficiency η on the domain thickness, which is in a qualitative agreement with the experiment. It is shown that planar domains on top of the nonpolar surface always enhance the value of η as compared with the bare surface.

  2. Bio-inspired direct patterning functional nanothin microlines: controllable liquid transfer.

    PubMed

    Wang, Qianbin; Meng, Qingan; Wang, Pengwei; Liu, Huan; Jiang, Lei

    2015-04-28

    Developing a general and low-cost strategy that enables direct patterning of microlines with nanometer thickness from versatile liquid-phase functional materials and precise positioning of them on various substrates remains a challenge. Herein, with inspiration from the oriental wisdom to control ink transfer by Chinese brushes, we developed a facile and general writing strategy to directly pattern various functional microlines with homogeneous distribution and nanometer-scale thickness. It is demonstrated that the width and thickness of the microlines could be well-controlled by tuning the writing method, providing guidance for the adaptation of this technique to various systems. It is also shown that various functional liquid-phase materials, such as quantum dots, small molecules, polymers, and suspensions of nanoparticles, could directly write on the substrates with intrinsic physicochemical properties well-preserved. Moreover, this technique enabled direct patterning of liquid-phase materials on certain microdomains, even in multiple layered style, thus a microdomain localized chemical reaction and the patterned surface chemical modification were enabled. This bio-inspired direct writing device will shed light on the template-free printing of various functional micropatterns, as well as the integrated functional microdevices. PMID:25845024

  3. Superhydrophobic film fabricated by controlled microphase separation of PEO-PLA mixture and its transparence property

    NASA Astrophysics Data System (ADS)

    Pi, Pihui; Mu, Wei; Fei, George; Deng, Yulin

    2013-05-01

    Instead of block copolymers that have been widely used in controlling thin film morphology, a mixture of two homopolymers has been used in this study to create desired nano- to microporous structure. By further modifying the nano-sized porous structured surface, a superhydrophobic surface was obtained. Experimentally, a chloroform solution containing a mixture of polylactic acid (PLA) and polyethylene oxide (PEO) was first coated on glass slides. Because of the dissimilarity of PLA and PEO, a microphase separation happened and the PEO formed microdomains in the coating layer during the film drying. Because PEO is water soluble but PLA is water-insoluble, the PEO microdomains could be washed out with water but PLA remained, resulted in a porous and rough PLA film. By two or three layer coating and washing, nano-sized roughness was obtained. A thin layer of fluorinated acrylic resin was further deposited on the rough surface. Because of the synergistic effect of surface roughness and hydrophobic, a superhydrophobicicity layer was obtained.

  4. Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions.

    PubMed

    Morita, Masamune; Onoe, Hiroaki; Yanagisawa, Miho; Ito, Hiroaki; Ichikawa, Masatoshi; Fujiwara, Kei; Saito, Hirohide; Takinoue, Masahiro

    2015-09-21

    We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2 μL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems. PMID:26212462

  5. Local structure of Rb{sub 2}Li{sub 4}(SeO{sub 4}){sub 3}{center_dot}2H{sub 2}O by the modeling of X-ray diffuse scattering - from average-structure to microdomain model

    SciTech Connect

    Komornicka, Dorota; Wolcyrz, Marek; Pietraszko, Adam

    2012-08-15

    Local structure of dirubidium tetralithium tris(selenate(VI)) dihydrate - Rb{sub 2}Li{sub 4}(SeO{sub 4}){sub 3}{center_dot} 2H{sub 2}O has been determined basing on the modeling of X-ray diffuse scattering. The origin of observed structured diffuse streaks is SeO{sub 4} tetrahedra switching between two alternative positions in two quasi-planar layers existing in each unit cell and formation of domains with specific SeO{sub 4} tetrahedra configuration locally fulfilling condition for C-centering in the 2a Multiplication-Sign 2b Multiplication-Sign c superstructure cell. The local structure solution is characterized by a uniform distribution of rather large domains (ca. thousand of unit cells) in two layers, but also monodomains can be taken into account. Inside a single domain SeO{sub 4} tetrahedra are ordered along ab-diagonal forming two-string ribbons. Inside the ribbons SeO{sub 4} and LiO{sub 4} tetrahedra share the oxygen corners, whereas ribbons are bound to each other by a net of hydrogen bonds and fastened by corner sharing SeO{sub 4} tetrahedra of the neighboring layers. - Graphical abstract: Experimental sections of the reciprocal space showing diffraction effects observed for RLSO. Bragg spots are visible on sections with integer indices (1 kl section - on the left), streaks - on sections with fractional ones (1.5 kl section - on the right). At the center: resulting local structure of the A package modeled as a microdomain: two-string ribbons of ordered oxygen-corners-sharing SeO{sub 4} and LiO{sub 4} terahedra extended along ab-diagonal are seen; ribbons are bound by hydrogen bonds (shown in pink); the multiplied 2a Multiplication-Sign 2b unit cell is shown. Highlights: Black-Right-Pointing-Pointer X-ray diffuse scattering in RLSO was registered and modeled. Black-Right-Pointing-Pointer The origin of diffuse streaks is SeO{sub 4} tetrahedra switching in two structure layers. Black-Right-Pointing-Pointer The local structure is characterized by a uniform

  6. Attractive membrane domains control lateral diffusion.

    PubMed

    Forstner, Martin B; Martin, Douglas S; Rückerl, Florian; Käs, Josef A; Selle, Carsten

    2008-05-01

    Lipid membranes play a fundamental role in vital cellular functions such as signal transduction. Many of these processes rely on lateral diffusion within the membrane, generally a complex fluid containing ordered microdomains. However, little attention has been paid to the alterations in transport dynamics of a diffusing species caused by long-range interactions with membrane domains. In this paper, we address the effect of such interactions on diffusive transport by studying lateral diffusion in a phase-separated Langmuir phospholipid monolayer via single-particle tracking. We find that attractive dipole-dipole interactions between condensed phase domains and diffusing probe beads lead to transient confinement at the phase boundaries, causing a transition from two- to one-dimensional diffusion. Using Brownian dynamics simulations, the long-term diffusion constant for such a system is found to have a sensitive, Boltzmann-like, dependence on the interaction strength. In addition, this interaction strength is shown to be a strong function of the ratio of domain to particle size. As similar interactions are expected in biological membranes, the modulation of diffusive transport dynamics by varying interaction strength and/or domain size may offer cells selective spatial and temporal control over signaling processes. PMID:18643101

  7. Dual-mode control of light by two-dimensional periodic structures realized in liquid-crystalline composite materials.

    PubMed

    De Sio, Luciano; Umeton, Cesare

    2010-08-15

    We report on the realization of a 2D refractive structure consisting of a polymer-liquid-crystal polymer slice grid. Nematic liquid crystal microdomains are confined inside well-sculptured elliptical cavities; experimental investigation shows that the liquid crystal director lies in the plane of the structure and its orientation follows a preferred direction. The sample exhibits both an electro-optical and an all-optical response owing to a small percentage of photosensitive azo dye included in the structure. A double external control of the two-dimensional grating efficiency can indeed be operated either by an optical pump beam or by the standard technique of applying a suitable external voltage to the sample. PMID:20717448

  8. How PI3K-derived lipids control cell division

    PubMed Central

    Campa, Carlo C.; Martini, Miriam; De Santis, Maria C.; Hirsch, Emilio

    2015-01-01

    To succeed in cell division, intense cytoskeletal and membrane remodeling are required to allow accurate chromosome segregation and cytoplasm partitioning. Spatial restriction of the actin dynamics and vesicle trafficking define the cell symmetry and equivalent membrane scission events, respectively. Protein complexes coordinating mitosis are recruited to membrane microdomains characterized by the presence of the phosphatidylinositol lipid members (PtdIns), like PtdIns(3,4,5)P3,PtdIns(4,5)P2, and PtdIns(3)P. These PtdIns represent a minor component of cell membranes, defining membrane domain identity, ultimately controlling cytoskeleton and membrane dynamics during mitosis. The coordinated presence of PtdIns(3,4,5)P3 at the cell poles and PtdIns(4,5)P2 at the cleavage furrow controls the polarity of the actin cytoskeleton leading to symmetrical cell division. In the endosomal compartment, the trafficking of PtdIns(3)P positive vesicles allows the recruitment of the protein machinery required for the abscission. PMID:26484344

  9. Galectin-4 Controls Intestinal Inflammation by Selective Regulation of Peripheral and Mucosal T Cell Apoptosis and Cell Cycle

    PubMed Central

    Paclik, Daniela; Danese, Silvio; Berndt, Uta; Wiedenmann, Bertram; Dignass, Axel; Sturm, Andreas

    2008-01-01

    Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease. PMID:18612433

  10. Lectin histochemistry reveals SNA as a prognostic carbohydrate-dependent probe for invasive ductal carcinoma of the breast: a clinicopathological and immunohistochemical auxiliary tool

    PubMed Central

    dos-Santos, Petra B; Zanetti, Juliana S; Vieira-de-Mello, Gabriela S; Rêgo, Moacyr BM; A, Alfredo Ribeiro-Silva; Beltrão, Eduardo Isidoro Carneiro

    2014-01-01

    Increased sialylation and β1,6-branched oligosaccharides has been associated with a variety of structural changes in cell surface carbohydrates, most notably in tumorigenesis. Lectins are defined as proteins that preferentially recognize and bind carbohydrate complexes protruding from glycolipids and glycoproteins. This interaction with carbohydrates can be as specific as the interaction between antigen and antibody. Due to this type of interaction lectins have been used as experimental auxiliary tools in histopathological diagnosis of cancer. This study was designed to evaluate the differential expression of sialic acids and β1,6-N-acetylglucosaminyltransferase V (MGAT5) in invasive (IDC) and in situ (DCIS) ductal carcinoma of the breast and its possible application as prognostic biomarkers. A possible transition between pre-malign and malign lesions was evaluated using DCIS samples. Biopsies were analyzed regarding the expression of MUC1, p53, Ki-67, estrogen receptor, progesterone receptor, HER-2 and MGAT5. α2,6-linked sialic acids residues recognized by SNA lectin was overexpressed in 33.3% of IDC samples and it was related with Ki-67 (p=0.042), PR (p=0.029), lymphnodes status (p=0.017) and death (p=0.011). Regarding survival analysis SNA was the only lectin able to correlate with specific-disease survival and disease-free survival (p=0.024 and p=0.041, respectively), besides, it presents itself as an independent variable by Cox Regression analysis (p= 0.004). Comparing IDC and DCIS cases, only SNA showed different staining pattern (p=0.034). The presence of sialic acids on tumor cell surface can be an indicative of poor prognosis and our study provides further evidence that SNA lectin can be used as a prognostic probe in IDC and DCIS patients. PMID:24966944

  11. High-Fat Diet Changes Hippocampal Apolipoprotein E (ApoE) in a Genotype- and Carbohydrate-Dependent Manner in Mice.

    PubMed

    Lane-Donovan, Courtney; Herz, Joachim

    2016-01-01

    Alzheimer's disease is a currently incurable neurodegenerative disease affecting millions of individuals worldwide. Risk factors for Alzheimer's disease include genetic risk factors, such as possession of ε4 allele of apolipoprotein E (ApoE4) over the risk-neutral ApoE3 allele, and lifestyle risk factors, such as diet and exercise. The intersection of these two sources of disease risk is not well understood. We investigated the impact of diet on ApoE levels by feeding wildtype, ApoE3, and ApoE4 targeted replacement (TR) mice with chow, high-fat, or ketogenic (high-fat, very-low-carbohydrate) diets. We found that high-fat diet affected both plasma and hippocampal levels of ApoE in an isoform-dependent manner, with high-fat diet causing a surprising reduction of hippocampal ApoE levels in ApoE3 TR mice. Conversely, the ketogenic diet had no effect on hippocampal ApoE. Our findings suggest that the use of dietary interventions to slow the progression AD should take ApoE genotype into consideration. PMID:26828652

  12. High-Fat Diet Changes Hippocampal Apolipoprotein E (ApoE) in a Genotype- and Carbohydrate-Dependent Manner in Mice

    PubMed Central

    Lane-Donovan, Courtney; Herz, Joachim

    2016-01-01

    Alzheimer’s disease is a currently incurable neurodegenerative disease affecting millions of individuals worldwide. Risk factors for Alzheimer’s disease include genetic risk factors, such as possession of ε4 allele of apolipoprotein E (ApoE4) over the risk-neutral ApoE3 allele, and lifestyle risk factors, such as diet and exercise. The intersection of these two sources of disease risk is not well understood. We investigated the impact of diet on ApoE levels by feeding wildtype, ApoE3, and ApoE4 targeted replacement (TR) mice with chow, high-fat, or ketogenic (high-fat, very-low-carbohydrate) diets. We found that high-fat diet affected both plasma and hippocampal levels of ApoE in an isoform-dependent manner, with high-fat diet causing a surprising reduction of hippocampal ApoE levels in ApoE3 TR mice. Conversely, the ketogenic diet had no effect on hippocampal ApoE. Our findings suggest that the use of dietary interventions to slow the progression AD should take ApoE genotype into consideration. PMID:26828652

  13. Probing Lipid Membrane Rafts (Microdomains) with Fluorescent Phospholipids

    NASA Astrophysics Data System (ADS)

    Gu, Yongwen; Mitchel, Drake

    2011-10-01

    Membrane rafts are enriched in sphingolipids and cholesterol, they exist in a more ordered state (the liquid-ordered phase; lo) than the bulk membrane (the liquid-disordered phase; ld). Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form membrane rafts over a wide range of molar ratios. We are examining the ability of two fluorescent probes, NBD linked to di-16:0 PE which partitions into the lo phase, and NBD linked to di-18:1 PE which partitions into the ld phase, to detect these two phases. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the size and stability of POPC/SPM/Chol membrane rafts. We report on the fluorescence lifetime and anisotropy decay dynamics of two fluorescent probes. Data were acquired via frequency-domain measurements from 5 to 250 MHz.

  14. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  15. Simple orientational control over cylindrical organic-inorganic block copolymer domains for etch mask applications.

    SciTech Connect

    Ramanathan, M.; Nettleton, E.; Darling, S. B.

    2009-02-01

    Bottom-up patterning methodologies, predicated on chemical self-assembly, have the potential to transcend limitations associated with more traditional lithographies. By controlling the domain orientation of a cylinder-forming organic-inorganic block copolymer, poly(styrene-block-ferrocenyldimethylsilane), it is possible to straightforwardly fabricate massive arrays of either nanoscale dots or wires out of a film composed of a wide variety of materials. In the work reported here, orientational control is achieved by manipulating the polymer film thickness in concert with the annealing treatment. For films much thinner than the equilibrium periodicity of the microdomains, the cylinders spontaneously orient themselves perpendicular to the substrate. Films with thickness close to the equilibrium periodicity exhibit the more common in-plane orientation following thermal annealing. Solvent annealing leads to an in-plane orientation for the full range of film thicknesses studied. As a demonstration of the effectiveness of this method, semiconductor substrates were patterned with arrays of posts and wires, respectively, using the same starting polymeric material as the etch mask. Compatibility of this polymer with various substrate materials is also demonstrated.

  16. A Glial K/Cl Transporter Controls Neuronal Receptive Ending Shape by Chloride Inhibition of an rGC.

    PubMed

    Singhvi, Aakanksha; Liu, Bingqian; Friedman, Christine J; Fong, Jennifer; Lu, Yun; Huang, Xin-Yun; Shaham, Shai

    2016-05-01

    Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here, we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes the NREs of 12 neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive ending microvilli, where it regulates K(+) and Cl(-) levels. We find that Cl(-) ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cyclic guanosine monophosphate (cGMP). High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function. PMID:27062922

  17. Tunable Surface Energy Interlayer Coating to Control the Phase Behavior of Block Copolymers in 2D Confinement

    NASA Astrophysics Data System (ADS)

    Hwang, Sungyoul; Kim, Youngkeol; Kwon, Dokyeong; Char, Kookheon

    There have been many studies to investigate the phase behavior of block copolymers (BCPs) in cylindrical confinement. In the nanometer scale 2D confinement, the phase behavior of BCPs is mainly dependent upon commensurability and interfacial interaction. However, most studies have focused only on the effects of commensurability on the microdomains of BCP. In this study, we employed organosilicate (OS) which has tunable surface energy upon adjusting curing temperature as interlayer to examine the phase behavior of BCPs as a function of interfacial energy. The OS interlayer was coated in the inner surface of anodized aluminum oxide (AAO) pores by template-wetting method and cured in a range of temperature to control the surface energy of the interlayer. Lamellae-forming poly(styrene-b-methyl methacrylate) (PS-b-PMMA) (SMA) in the melt was injected into the OS-coated AAO pores by capillary forces. With the detailed analysis, we note that the self-assembly of SMA within 2D confinement is competitively affected by both entropic and enthalpic effects as the contact interfacial energy is varied. Simply by controlling the curing temperature of the OS interlayer, various morphologies arising from both preferential and neutral wetting were identified.

  18. The STIM1-Orai1 pathway of store-operated Ca2+ entry controls the checkpoint in cell cycle G1/S transition

    PubMed Central

    Chen, Yun-Wen; Chen, Yih-Fung; Chen, Ying-Ting; Chiu, Wen-Tai; Shen, Meng-Ru

    2016-01-01

    Ca2+ signaling is important to trigger the cell cycle progression, while it remains elusive in the regulatory mechanisms. Here we show that store-operated Ca2+ entry (SOCE), mediated by the interaction between STIM1 (an endoplasmic reticulum Ca2+ sensor) and Orai1 (a cell membrane pore structure), controls the specific checkpoint of cell cycle. The fluctuating SOCE activity during cell cycle progression is universal in different cell types, in which SOCE is upregulated in G1/S transition and downregulated from S to G2/M transition. Pharmacological or siRNA inhibition of STIM1-Orai1 pathway of SOCE inhibits the phosphorylation of CDK2 and upregulates the expression of cyclin E, resulting in autophagy accompanied with cell cycle arrest in G1/S transition. The subsequently transient expression of STIM1 cDNA in STIM1−/− MEF rescues the phosphorylation and nuclear translocation of CDK2, suggesting that STIM1-mediated SOCE activation directly regulates CDK2 activity. Opposite to the important role of SOCE in controlling G1/S transition, the downregulated SOCE is a passive phenomenon from S to G2/M transition. This study uncovers SOCE-mediated Ca2+ microdomain that is the molecular basis for the Ca2+ sensitivity controlling G1/S transition. PMID:26917047

  19. Control of Hepatic Nuclear Superoxide Production by Glucose 6-Phosphate Dehydrogenase and NADPH Oxidase-4*

    PubMed Central

    Spencer, Netanya Y.; Yan, Ziying; Boudreau, Ryan L.; Zhang, Yulong; Luo, Meihui; Li, Qiang; Tian, Xin; Shah, Ajay M.; Davisson, Robin L.; Davidson, Beverly; Banfi, Botond; Engelhardt, John F.

    2011-01-01

    Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O2⨪); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22phox, and p47phox) contribute to nuclear O2⨪ production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O2⨪ production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O2⨪ predominantly localized to the perinuclear space. Interestingly, NADP+ and G6P also induced nuclear O2⨪ production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O2⨪ in response to NADP+ and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O2⨪ production by NOX4. PMID:21212270

  20. Dream controller

    DOEpatents

    Cheng, George Shu-Xing; Mulkey, Steven L; Wang, Qiang; Chow, Andrew J

    2013-11-26

    A method and apparatus for intelligently controlling continuous process variables. A Dream Controller comprises an Intelligent Engine mechanism and a number of Model-Free Adaptive (MFA) controllers, each of which is suitable to control a process with specific behaviors. The Intelligent Engine can automatically select the appropriate MFA controller and its parameters so that the Dream Controller can be easily used by people with limited control experience and those who do not have the time to commission, tune, and maintain automatic controllers.

  1. Birth Control

    MedlinePlus

    Birth control, also known as contraception, is designed to prevent pregnancy. Birth control methods may work in a number of different ... eggs that could be fertilized. Types include birth control pills, patches, shots, vaginal rings, and emergency contraceptive ...

  2. DEVICE CONTROLLER, CAMERA CONTROL

    Energy Science and Technology Software Center (ESTSC)

    1998-07-20

    This is a C++ application that is the server for the cameral control system. Devserv drives serial devices, such as cameras and videoswitchers used in a videoconference, upon request from a client such as the camxfgbfbx ccint program. cc Deverv listens on UPD ports for clients to make network contractions. After a client connects and sends a request to control a device (such as to pan,tilt, or zooma camera or do picture-in-picture with a videoswitcher),more » devserv formats the request into an RS232 message appropriate for the device and sends this message over the serial port to which the device is connected. Devserv then reads the reply from the device from the serial port to which the device is connected. Devserv then reads the reply from the device from the serial port and then formats and sends via multicast a status message. In addition, devserv periodically multicasts status or description messages so that all clients connected to the multicast channel know what devices are supported and their ranges of motion and the current position. The software design employs a class hierarchy such that an abstract base class for devices can be subclassed into classes for various device categories(e.g. sonyevid30, cononvco4, panasonicwjmx50, etc.). which are further subclassed into classes for various device categories. The devices currently supported are the Sony evi-D30, Canon, VCC1, Canon VCC3, and Canon VCC4 cameras and the Panasonic WJ-MX50 videoswitcher. However, developers can extend the class hierarchy to support other devices.« less

  3. DEVICE CONTROLLER, CAMERA CONTROL

    SciTech Connect

    Perry, Marcia

    1998-07-20

    This is a C++ application that is the server for the cameral control system. Devserv drives serial devices, such as cameras and videoswitchers used in a videoconference, upon request from a client such as the camxfgbfbx ccint program. cc Deverv listens on UPD ports for clients to make network contractions. After a client connects and sends a request to control a device (such as to pan,tilt, or zooma camera or do picture-in-picture with a videoswitcher), devserv formats the request into an RS232 message appropriate for the device and sends this message over the serial port to which the device is connected. Devserv then reads the reply from the device from the serial port to which the device is connected. Devserv then reads the reply from the device from the serial port and then formats and sends via multicast a status message. In addition, devserv periodically multicasts status or description messages so that all clients connected to the multicast channel know what devices are supported and their ranges of motion and the current position. The software design employs a class hierarchy such that an abstract base class for devices can be subclassed into classes for various device categories(e.g. sonyevid30, cononvco4, panasonicwjmx50, etc.). which are further subclassed into classes for various device categories. The devices currently supported are the Sony evi-D30, Canon, VCC1, Canon VCC3, and Canon VCC4 cameras and the Panasonic WJ-MX50 videoswitcher. However, developers can extend the class hierarchy to support other devices.

  4. Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

    PubMed

    Plattner, Helmut; Verkhratsky, Alexei

    2016-08-01

    From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally

  5. Rodent Control

    ERIC Educational Resources Information Center

    Indian Journal of Adult Education, 1975

    1975-01-01

    Strategies for rodent control in crop fields, threshing yards, and rural residential areas are presented together with an operational plan for implementing a program for rodent control at the national level. Training personnel in rodent control procedures and procedures for educating the public in the necessity for control are covered. (EC)

  6. Propulsion controls

    NASA Technical Reports Server (NTRS)

    Harkney, R. D.

    1980-01-01

    Increased system requirements and functional integration with the aircraft have placed an increased demand on control system capability and reliability. To provide these at an affordable cost and weight and because of the rapid advances in electronic technology, hydromechanical systems are being phased out in favor of digital electronic systems. The transition is expected to be orderly from electronic trimming of hydromechanical controls to full authority digital electronic control. Future propulsion system controls will be highly reliable full authority digital electronic with selected component and circuit redundancy to provide the required safety and reliability. Redundancy may include a complete backup control of a different technology for single engine applications. The propulsion control will be required to communicate rapidly with the various flight and fire control avionics as part of an integrated control concept.

  7. Weight Control

    MedlinePlus

    ... obese. Achieving a healthy weight can help you control your cholesterol, blood pressure and blood sugar. It ... use more calories than you eat. A weight-control strategy might include Choosing low-fat, low-calorie ...

  8. Controlling Fertility.

    ERIC Educational Resources Information Center

    Donnay, France

    1991-01-01

    Recent developments in fertility control are presented in relation to the global demographic situation. Discussion focuses on changes in scientific knowledge and concepts that have shifted the focus from birth control to planned parenthood to the notion of controlled fertility. The place of family planning programs, including their socioeconomic…

  9. Birth Control

    MedlinePlus

    ... to have sex makes sense Talking to your parents about sex Deciding about sex Birth control Types of birth control Could I get pregnant ... not planned. Some young people are afraid their parents will find out they’re having sex. If you get birth control from a doctor, ask about keeping the information ...

  10. Restructurable Controls

    NASA Technical Reports Server (NTRS)

    Montoya, R. J. (Compiler); Howell, W. E. (Compiler); Bundick, W. T. (Compiler); Ostroff, A. J. (Compiler); Hueschen, R. M. (Compiler); Belcastro, C. M. (Compiler)

    1983-01-01

    Restructurable control system theory, robust reconfiguration for high reliability and survivability for advanced aircraft, restructurable controls problem definition and research, experimentation, system identification methods applied to aircraft, a self-repairing digital flight control system, and state-of-the-art theory application are addressed.

  11. Controlled drugs.

    PubMed

    2016-05-18

    Essential facts Controlled drugs are defined and governed by the Misuse of Drugs Act 1971 and associated regulations. Examples of controlled drugs include morphine, pethidine and methadone. Since 2012, appropriately qualified nurses and midwives can prescribe controlled drugs for medical conditions within their competence. There are some exceptions when treating addiction. PMID:27191427

  12. Weed control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Uncontrolled weeds in pepper can result in the total loss of the crop, or make harvest not economically feasible. Weed control is a major challenge in conventional and organic production systems. Agricultural weed control costs the U.S. economy more than the cost of insect and disease control comb...

  13. Characteristics of microdomains and microdomain patterns recorded by electron beam irradiation on Y-cut LiNbO{sub 3} crystals

    SciTech Connect

    Kokhanchik, L. S.; Gainutdinov, R. V.; Volk, T. R.; Lavrov, S. D.

    2015-08-21

    We present the results of investigations of planar domain patterns (isolated domains and domain gratings) fabricated by irradiation of the nonpolar Y-surface of LiNbO{sub 3} crystals by an electron beam (EB) incident normally onto the surface. The EB recorded domains were investigated using atomic force microscopy, confocal second harmonic generation microscopy, and chemical etching as an auxiliary method. The dependence of the domain characteristics on irradiation conditions (acceleration voltage U, EB current I, and irradiation time t{sub irr}) were determined. The length L{sub d} of both isolated domains and domain gratings along the polar axis Z grows linearly with t{sub irr} (at U, I = const) with no tending to saturation. The plots L{sub d}(t{sub irr}) obtained for U = 10 and 15 kV are practically identical, whereas the values of L{sub d} for U = 5 kV are essentially lower. The domain thickness T{sub d} along the Y-direction, i.e., the depth of the switched layer grows with acceleration voltage U. These results are discussed in terms of space-charge fields formation arising under EB irradiation of insulators. The linearity of L{sub d}(t{sub irr}) is accounted for by the frontal domain growth via the viscous friction law. The experimental dependence of T{sub d} on U supports the suggestion that the domain thickness is determined by the penetration depth R{sub e} of primary electrons, which in turn is governed by U. The difference in L{sub d}(t{sub irr}) plots for different U is accounted for by different electron emission σ. Indirect evidences of a defect structure modification in a thin surface layer with respect to the crystal bulk are obtained.

  14. Voltage Controller

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Power Efficiency Corporation, specifically formed to manufacture and develop products from NASA technology, has a license to a three-phase power factor controller originally developed by Frank Nola, an engineer at Marshall Space Flight Center. Power Efficiency and two major distributors, Performance Control and Edison Power Technologies, use the electronic control boards to assemble three different motor controllers: Power Commander, Performance Controller, and Energy Master. The company Power Factor Controller reduces excessive energy waste in AC induction motors. It is used in industries and applications where motors operate under variable loads, including elevators and escalators, machine tools, intake and exhaust fans, oil wells, conveyors, pumps, die casting, and compressors. Customer lists include companies such as May Department Stores, Caesars Atlantic City, Ford Motors, and American Axle.

  15. Control Software

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Real-Time Innovations, Inc. (RTI) collaborated with Ames Research Center, the Jet Propulsion Laboratory and Stanford University to leverage NASA research to produce ControlShell software. RTI is the first "graduate" of Ames Research Center's Technology Commercialization Center. The ControlShell system was used extensively on a cooperative project to enhance the capabilities of a Russian-built Marsokhod rover being evaluated for eventual flight to Mars. RTI's ControlShell is complex, real-time command and control software, capable of processing information and controlling mechanical devices. One ControlShell tool is StethoScope. As a real-time data collection and display tool, StethoScope allows a user to see how a program is running without changing its execution. RTI has successfully applied its software savvy in other arenas, such as telecommunications, networking, video editing, semiconductor manufacturing, automobile systems, and medical imaging.

  16. Attitude Control

    NASA Technical Reports Server (NTRS)

    1997-01-01

    A Small Business Innovation Research (SBIR) contract to ITHACO, Inc. satisfied a Goddard Space Flight Center demand for a low cost altitude control system for small spacecraft. The SBIR-sponsored work resulted in the T-Wheel, built specifically for altitude control of small and medium-sized spacecraft. Another product, the T-SCANWHEEL, reduces overall system cost, minimizes mass and power and enhances reliability with a mixture of altitude control and control capacity. Additionally, the Type E Wheel is built for use on medium to large spacecraft. Through July 1996, ITHACO had delivered or was under contract for 95 T-Wheel, T-SCANWHEEL, and Type E Wheel units.

  17. Orientation Control in Thin Films of a High-χ Block Copolymer with a Surface Active Embedded Neutral Layer.

    PubMed

    Zhang, Jieqian; Clark, Michael B; Wu, Chunyi; Li, Mingqi; Trefonas, Peter; Hustad, Phillip D

    2016-01-13

    Directed self-assembly (DSA) of block copolymers (BCPs) is an attractive advanced patterning technology being considered for future integrated circuit manufacturing. By controlling interfacial interactions, self-assembled microdomains in thin films of polystyrene-block-poly(methyl methacrylate), PS-b-PMMA, can be oriented perpendicular to surfaces to form line/space or hole patterns. However, its relatively weak Flory interaction parameter, χ, limits its capability to pattern sub-10 nm features. Many BCPs with higher interaction parameters are capable of forming smaller features, but these "high-χ" BCPs typically have an imbalance in surface energy between the respective blocks that make it difficult to achieve the required perpendicular orientation. To address this challenge, we devised a polymeric surface active additive mixed into the BCP solution, referred to as an embedded neutral layer (ENL), which segregates to the top of the BCP film during casting and annealing and balances the surface tensions at the top of the thin film. The additive comprises a second BCP with a "neutral block" designed to provide matched surface tensions with the respective polymers of the main BCP and a "surface anchoring block" with very low surface energy that drives the material to the air interface during spin-casting and annealing. The surface anchoring block allows the film to be annealed above the glass transition temperature of the two materials without intermixing of the two components. DSA was also demonstrated with this embedded neutral top layer formulation on a chemical patterned template using a single step coat and simple thermal annealing. This ENL technology holds promise to enable the use of high-χ BCPs in advanced patterning applications. PMID:26682931

  18. Environmental Controls.

    ERIC Educational Resources Information Center

    Schneiderman, Helen, Ed.

    1994-01-01

    Environmental control units, or ECUs, are devices or systems which allow for alternate access to electronic or electrical devices and those objects, like draperies and doors, which may be adapted for use with electricity. Such devices offer the person with a mobility limitation the opportunity to control his or her environment, thus enhancing the…

  19. Pressure Controller

    NASA Astrophysics Data System (ADS)

    1981-01-01

    EPIC is Electronic Pressure Indicating Controller produced by North American Manufacturing Company. It is a high-sensitivity device for improving combustion efficiency in industrial furnaces that interprets a signal from a pressure transducer on a furnace and regulates furnace pressure accordingly. A controller can provide savings of from five to 25 percent of an industrial user's annual furnace fuel bill.

  20. Contamination control

    SciTech Connect

    Jackson, L.C.

    1983-11-01

    An evaluation showed that fluoropolymer plastic squeeze bottles can replace polyethylene bottles when used for in-process cleaning. Fluoropolymer plastic squeeze bottles do not contaminate solvents stored in the bottles as polyethylene bottles do. In addition, a limited survey of industrial practices regarding aerosol spray container control showed containers are being controlled without inconveniencing production.

  1. Control Charts

    NASA Astrophysics Data System (ADS)

    Koch, Michael; Gluschke, Michael

    Control Charts may be the most powerful tool to demonstrate and to assure quality in chemical measurements. Therefore they are widely used in all kinds of laboratories and it is hard to imagine quality management systems in laboratories without control charts.

  2. Power Controller

    NASA Technical Reports Server (NTRS)

    1983-01-01

    The power factor controller (PFC) senses shifts in the relationship between voltage and current, and matches them with a motor's need. This prevents waste as motors do not need a high voltage when they are not operating at full load conditions. PFC is manufactured by Nordic Controls Company, among others, and has proved extremely cost effective.

  3. Control Theory.

    ERIC Educational Resources Information Center

    Toso, Robert B.

    2000-01-01

    Inspired by William Glasser's Reality Therapy ideas, Control Theory (CT) is a disciplinary approach that stresses people's ability to control only their own behavior, based on internal motivations to satisfy five basic needs. At one North Dakota high school, CT-trained teachers are the program's best recruiters. (MLH)

  4. Controller for thermostatically controlled loads

    DOEpatents

    Lu, Ning; Zhang, Yu; Du, Pengwei; Makarov, Yuri V.

    2016-06-07

    A system and method of controlling aggregated thermostatically controlled appliances (TCAs) for demand response is disclosed. A targeted load profile is formulated and a forecasted load profile is generated. The TCAs within an "on" or "off" control group are prioritized based on their operating temperatures. The "on" or "off" status of the TCAs is determined. Command signals are sent to turn on or turn off the TCAs.

  5. Controlling chimeras

    NASA Astrophysics Data System (ADS)

    Bick, Christian; Martens, Erik A.

    2015-03-01

    Coupled phase oscillators model a variety of dynamical phenomena in nature and technological applications. Non-local coupling gives rise to chimera states which are characterized by a distinct part of phase-synchronized oscillators while the remaining ones move incoherently. Here, we apply the idea of control to chimera states: using gradient dynamics to exploit drift of a chimera, it will attain any desired target position. Through control, chimera states become functionally relevant; for example, the controlled position of localized synchrony may encode information and perform computations. Since functional aspects are crucial in (neuro-)biology and technology, the localized synchronization of a chimera state becomes accessible to develop novel applications. Based on gradient dynamics, our control strategy applies to any suitable observable and can be generalized to arbitrary dimensions. Thus, the applicability of chimera control goes beyond chimera states in non-locally coupled systems.

  6. CONTROL ROD

    DOEpatents

    Zinn, W.H.; Ross, H.V.

    1958-11-18

    A control rod is described for a nuclear reactor. In certaln reactor designs it becomes desirable to use a control rod having great width but relatively llttle thickness. This patent is addressed to such a need. The neutron absorbing material is inserted in a triangular tube, leaving volds between the circular insert and the corners of the triangular tube. The material is positioned within the tube by the use of dummy spacers to achleve the desired absorption pattern, then the ends of the tubes are sealed with suitable plugs. The tubes may be welded or soldered together to form two flat surfaces of any desired width, and covered with sheetmetal to protect the tubes from damage. This design provides a control member that will not distort under the action of outside forces or be ruptured by gases generated within the jacketed control member.

  7. Infection Control

    MedlinePlus

    ... lost because of the spread of infections in hospitals. Health care workers can take steps to prevent the spread of infectious diseases. These steps are part of infection control. Proper hand washing is the most effective way ...

  8. Drug Control

    ERIC Educational Resources Information Center

    Leviton, Harvey S.

    1975-01-01

    This article attempts to assemble pertinent information about the drug problem, particularily marihuana. It also focuses on the need for an educational program for drug control with the public schools as the main arena. (Author/HMV)

  9. Under Control.

    ERIC Educational Resources Information Center

    Henry, Rich

    2001-01-01

    Offers advice on how school administrators can properly plan and monitor school construction projects to contain costs. Cost control tips discussed include project scope definition, contract bidding and awarding practice, and project management techniques. (GR)

  10. REACTOR CONTROL

    DOEpatents

    Fortescue, P.; Nicoll, D.

    1962-04-24

    A control system employed with a high pressure gas cooled reactor in which a control rod is positioned for upward and downward movement into the neutron field from a position beneath the reactor is described. The control rod is positioned by a coupled piston cylinder releasably coupled to a power drive means and the pressurized coolant is directed against the lower side of the piston. The coolant pressure is offset by a higher fiuid pressure applied to the upper surface of the piston and means are provided for releasing the higher pressure on the upper side of the piston so that the pressure of the coolant drives the piston upwardly, forcing the coupled control rod into the ncutron field of the reactor. (AEC)

  11. Fuel control

    SciTech Connect

    Charrier, M.G.

    1986-08-19

    This patent describes a gas turbine engine control which responds to a compressor discharge pressure (CDP) and a compressor bleed pressure (CBP). The improvement described here consists of: (a) sensing means for sensing whether the aircraft is on the ground and (b) means coupled to the sensing means for artificially increasing the ratio of CBP/CDP which is sensed by the control, during engine startup, while the aircraft is on the ground.

  12. Statins Attenuate Helicobacter pylori CagA Translocation and Reduce Incidence of Gastric Cancer: In Vitro and Population-Based Case-Control Studies

    PubMed Central

    Hsu, Yuan-Man; Lin, Cheng-Li; Chen, Yu-An; Feng, Chun-Lung; Chen, Chih-Jung; Kao, Min-Chuan; Lai, Chih-Ho; Kao, Chia-Hung

    2016-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide. The correlation of Helicobacter pylori and the etiology of gastric cancer was substantially certain. Cholesterol-rich microdomains (also called lipid rafts), which provide platforms for signaling, are associated with H. pylori-induced pathogenesis leading to gastric cancer. Patients who have been prescribed statins, inhibitors of 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase, have exhibited a reduced risk of several types of cancer. However, no studies have addressed the effect of statins on H. pylori-associated gastric cancer from the antineoplastic perspective. In this study, we showed that treatment of gastric epithelial cells with simvastatin reduced the level of cellular cholesterol and led to attenuation of translocation and phosphorylation of H. pylori cytotoxin-associated gene A (CagA), which is recognized as a major determinant of gastric cancer development. Additionally, a nationwide case-control study based on data from the Taiwanese National Health Insurance Research Database (NHIRD) was conducted. A population-based case-control study revealed that patients who used simvastatin exhibited a significantly reduced risk of gastric cancer (adjusted odds ratio (OR) = 0.76, 95% confidence interval (CI) = 0.70–0.83). In patients exhibiting H. pylori infection who were prescribed simvastatin, the adjusted OR for gastric cancer was 0.25 (95% CI = 0.12–0.50). Our results combined an in vitro study with a nationwide population analysis reveal that statin use might be a feasible approach to prevent H. pylori-associated gastric cancer. PMID:26730715

  13. Grain Rotation in Ion-Complexed Symmetric Diblock Copolymer Thin Films under an Electric Field

    SciTech Connect

    Wang,J.; Leiston-Belanger, J.; Sievert, J.; Russell, T.

    2006-01-01

    In symmetric polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) diblock copolymer thin films, lithium-PMMA complexes were formed with the addition of lithium chloride (LiCl), significantly increasing both {chi} and dielectric constant. These led to a transition in the kinetic pathway of the orientation of lamellar microdomains under an applied electric field from a disruption and re-formation of the microdomains to a grain rotation mediated by movement of defects. By controlling the number of lithium-PMMA complexes, the microdomain alignment is possibly regulated in PS-b-PMMA copolymer thin films.

  14. COPD - control drugs

    MedlinePlus

    Chronic obstructive pulmonary disease - control drugs; Bronchodilators - COPD - control drugs; Beta agonist inhaler - COPD - control drugs; Anticholinergic inhaler - COPD - control drugs; Long-acting inhaler - COPD - control drugs; ...

  15. Controlling Stress

    ERIC Educational Resources Information Center

    Kelehear, Zach

    2004-01-01

    Stress occurs among all groups in a school community and can affect morale, performance, and leadership ability. This article discusses how educators must deal with and control stress. Principals must address their own stress to create a healthy school culture. Moreover, the author presents two survival strategies that can provide support and…

  16. Controlling chaos

    SciTech Connect

    Ott, E.; Spano, M.

    1996-06-01

    The orbital complexity and exponential sensitivity of chaotic systems has the consequence that such systems offer the possibility of being feedback controlled by use of only small perturbations. The potential consequences of this recent realization are being investigated in a broad range of applications. {copyright} {ital 1996 American Institute of Physics.}

  17. Motor Controllers

    NASA Technical Reports Server (NTRS)

    1984-01-01

    Kollmorgen Corporation's Mermaid II two person submersible is propeller-driven by a system of five DC brushless motors with new electronic controllers that originated in work performed in a NASA/DOE project managed by Lewis Research Center. A key feature of the system is electric commutation rather than mechanical commutation for converting AC current to DC.

  18. Controlling turbulence

    NASA Astrophysics Data System (ADS)

    Kühnen, Jakob; Hof, Björn

    2015-11-01

    We show that a simple modification of the velocity profile in a pipe can lead to a complete collapse of turbulence and the flow fully relaminarises. The annihilation of turbulence is achieved by a steady manipulation of the streamwise velocity component alone, greatly reducing control efforts. Several different control techniques are presented: one with a local modification of the flow profile by means of a stationary obstacle, one employing a nozzle injecting fluid through a small gap at the pipe wall and one with a moving wall, where a part of the pipe is shifted in the streamwise direction. All control techniques act on the flow such that the streamwise velocity profile becomes more flat and turbulence gradually grows faint and disappears. In a smooth straight pipe the flow remains laminar downstream of the control. Hence a reduction in skin friction by a factor of 8 and more can be accomplished. Stereoscopic PIV-measurements and movies of the development of the flow during relaminarisation are presented.

  19. Birth Control

    MedlinePlus

    ... your health, frequency of sexual activity, number of sexual partners and desire to have children in the future. Your health care provider can help you select the best form of birth control for you. NIH: National Institute of Child Health and Human Development

  20. Autonomous control

    NASA Technical Reports Server (NTRS)

    Brown, Barbara

    1990-01-01

    KSC has been developing the Knowledge-Based Autonomous Test Engineer (KATE), which is a tool for performing automated monitoring, diagnosis, and control of electromechanical devices. KATE employs artificial intelligence computing techniques to perform these functions. The KATE system consists of a generic shell and a knowledge base. The KATE shell is the portion of the system which performs the monitoring, diagnosis, and control functions. It is generic in the sense that it is application independent. This means that the monitoring activity, for instance, will be performed with the same algorithms regardless of the particular physical device being used. The knowledge base is the portion of the system which contains specific functional and behavorial information about the physical device KATE is working with. Work is nearing completion on a project at KSC to interface a Texas Instruments Explorer running a LISP version of KATE with a Generic Checkout System (GCS) test-bed to control a physical simulation of a shuttle tanking system (humorously called the Red Wagon because of its color and mobility). The Autonomous Control System (ACS) project supplements and extends the KATE/GCS project by adding three other major activities. The activities include: porting KATE from the Texas Instruments Explorer machine to an Intel 80386-based UNIX workstation in the LISP language; rewriting KATE as necessary to run on the same 80386 workstation but in the Ada language; and investigating software and techniques to translate ANSI Standard Common LISP to Mil Standard Ada. Primary goals of this task are as follows: (1) establish the advantages of using expert systems to provide intelligent autonomous software for Space Station Freedom applications; (2) determine the feasibility of using Ada as the run-time environment for model-based expert systems; (3) provide insight into the advantages and disadvantagesof using LISP or Ada in the run-time environment for expert systems; and (4

  1. REACTOR CONTROL

    DOEpatents

    Ruano, W.J.

    1957-12-10

    This patent relates to nuclear reactors of the type which utilize elongited rod type fuel elements immersed in a liquid moderator and shows a design whereby control of the chain reaction is obtained by varying the amount of moderator or reflector material. A central tank for containing liquid moderator and fuel elements immersed therein is disposed within a surrounding outer tank providing an annular space between the two tanks. This annular space is filled with liquid moderator which functions as a reflector to reflect neutrons back into the central reactor tank to increase the reproduction ratio. Means are provided for circulating and cooling the moderator material in both tanks and additional means are provided for controlling separately the volume of moderator in each tank, which latter means may be operated automatically by a neutron density monitoring device. The patent also shows an arrangement for controlling the chain reaction by injecting and varying an amount of poisoning material in the moderator used in the reflector portion of the reactor.

  2. Glucose control.

    PubMed

    Preiser, Jean-Charles

    2013-01-01

    Stress-related hyperglycemia is a common finding in acutely ill patients, and is related to the severity and outcome of the critical illness. The pathophysiology of stress hyperglycemia includes hormonal and neural signals, leading to increased production of glucose by the liver and peripheral insulin resistance mediated by the translocation of transmembrane glucose transporters. In one pioneering study, tight glycemic control by intensive insulin therapy in critically ill patients was associated with improved survival. However, this major finding was not confirmed in several other prospective randomized controlled trials. The reasons underlying the discrepancy between the first and the subsequent studies could include nutritional strategy (amount of calories provided, use of parenteral nutrition), case-mix, potential differences in the optimal blood glucose level (BG) in different types of patients, hypoglycemia and its correction, and the magnitude of glucose variability. Therefore, an improved understanding of the physiology and pathophysiology of glycemic regulation during acute illness is needed. Safe and effective glucose control will need improvement in the definition of optimal BG and in the measurement techniques, perhaps including continuous monitoring of insulin algorithms and closed-loop systems. PMID:23075589

  3. Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis

    PubMed Central

    Jenkins, Paul M.; He, Meng; Bennett, Vann

    2015-01-01

    Current physical models for plasma membranes emphasize dynamic 10- to 300-nm compartments at thermodynamic equilibrium but subject to thermal fluctuations. However, epithelial lateral membranes contain micrometer-sized domains defined by an underlying membrane skeleton composed of spectrin and its partner ankyrin-G. We demonstrate that these spectrin/ankyrin-G domains exhibit local microtubule-dependent movement on a time scale of minutes and encounter most of the lateral membranes within an hour. Spectrin/ankyrin-G domains exclude clathrin and clathrin-dependent cargo, and inhibit both receptor-mediated and bulk endocytosis. Moreover, inhibition of endocytosis fully restores lateral membrane height in spectrin- or ankyrin-G–depleted cells. These findings support a non-equilibrium cellular-scale model for epithelial lateral membranes, where spectrin/ankyrin-G domains actively patrol the plasma membrane, analogous to “window washers,” and promote columnar morphology by blocking membrane uptake. PMID:26523289

  4. Clarin-1, Encoded by the Usher Syndrome III Causative Gene, Forms a Membranous Microdomain

    PubMed Central

    Tian, Guilian; Zhou, Yun; Hajkova, Dagmar; Miyagi, Masaru; Dinculescu, Astra; Hauswirth, William W.; Palczewski, Krzysztof; Geng, Ruishuang; Alagramam, Kumar N.; Isosomppi, Juha; Sankila, Eeva-Marja; Flannery, John G.; Imanishi, Yoshikazu

    2009-01-01

    Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems. When expressed in HEK293 cells, clarin-1 localized to the plasma membrane and concentrated in low density compartments distinct from lipid rafts. Clarin-1 reorganized actin filament structures and induced lamellipodia. This actin-reorganizing function was absent in the modified protein encoded by the most prevalent North American Usher syndrome III mutation, the N48K form of clarin-1 deficient in N-linked glycosylation. Proteomics analyses revealed a number of clarin-1-interacting proteins involved in cell-cell adhesion, focal adhesions, cell migration, tight junctions, and regulation of the actin cytoskeleton. Consistent with the hypothesized role of clarin-1 in actin organization, F-actin-enriched stereocilia of auditory hair cells evidenced structural disorganization in Clrn1−/− mice. These observations suggest a possible role for clarin-1 in the regulation and homeostasis of actin filaments, and link clarin-1 to the interactive network of Usher syndrome gene products. PMID:19423712

  5. Influence of irradiation-induced disorder on the Peierls transition in TTF-TCNQ microdomains

    NASA Astrophysics Data System (ADS)

    Solovyeva, Vita; Cmyrev, Anastasia; Sachser, Roland; Reith, Heiko; Huth, Michael

    2011-09-01

    The combined influence of electron irradiation-induced defects, substrate-induced strain and finite size effects on the electronic transport properties of individual micron-sized thin film growth domains of the organic charge transfer compound tetrathiafulvalene- tetracyanoquinodimethane (TTF-TCNQ) have been studied. The TTF-TCNQ domains have been isolated and electrically contacted by focused ion beam etching and focused ion and electron-beam-induced deposition, respectively. This allowed us to measure the temperature-dependent resistivity and the current-voltage characteristics of individual domains. The dependence of the resistivity on temperature follows a variable-range hopping behaviour which shows a crossover of the exponents as the Peierls transition is approached. The low temperature behaviour is analysed within the segmented rod model of Fogler, Teber and Shklovskii which was developed for charge-ordered quasi one-dimensional electron crystals (Fogler et al 2004 Phys. Rev. B 69 035413). The effect of substrate-induced biaxial strain on the Peierls transition temperature is discussed with regard to its interplay with the defect-induced changes.

  6. Ca(2+) microdomains, NAADP and type 1 ryanodine receptor in cell activation.

    PubMed

    Guse, Andreas H; Wolf, Insa M A

    2016-06-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+) mobilizing second messenger that belongs to the superfamily of regulatory adenine nucleotides. Though NAADP has been known since 20years, several aspects of its metabolism and molecular mode of action are still under discussion. Though the importance of the type 1 ryanodine receptor was discovered and published already in 2002 Hohenegger et al. (2002 Oct 15) , recent data re-emphasize these original findings in pancreatic acinar cells and in T-lymphocytes. Here we review recent developments in NAADP formation and metabolism, putative target Ca(2+) channels for NAADP with special emphasis on the type 1 ryanodine receptor, and NAADP binding proteins. The latter are basis for a unifying hypothesis for NAADP action. Finally, the role of NAADP in T cell Ca(2+) signaling and activation is discussed. This article is part of a Special Issue entitled: Calcium and Cell Fate . Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen. PMID:26804481

  7. Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

    PubMed Central

    Marks, David L.; Bittman, Robert

    2014-01-01

    Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple “microdomains” of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-d-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane. PMID:18820942

  8. Dopaminergic and glutamatergic microdomains within a subset of rodent mesoaccumbens axons

    PubMed Central

    Zhang, Shiliang; Qi, Jia; Li, Xueping; Wang, Hui-Ling; Britt, Jonathan P.; Hoffman, Alexander F.; Bonci, Antonello; Lupica, Carl R.; Morales, Marisela

    2015-01-01

    Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism of co-release of dopamine and glutamate is unclear, and the co-release by mesoaccumbens fibers has not been documented. By electron microcopy we showed that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon-segments continuous with axon-terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change segregation of the two vesicular types, suggesting highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2-vesicles make asymmetric synapses; commonly associated with excitatory signaling. By optogenetics, we showed release of dopamine and glutamate from mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate although can be released from the same axons; they are not normally released at the same site or from the same synaptic vesicles. PMID:25664911

  9. Segregation of Platelet Aggregatory and Procoagulant Microdomains in Thrombus Formation Regulation by Transient Integrin Activation

    PubMed Central

    Munnix, Imke C.A.; Kuijpers, Marijke J.E.; Auger, Jocelyn; Thomassen, Christella M.L.G.D.; Panizzi, Peter; van Zandvoort, Marc A.M.; Rosing, Jan; Bock, Paul E.; Watson, Steve P.; Heemskerk, Johan W.M.

    2008-01-01

    Objective Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo. Methods and Results High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine. The latter platelet population had inactivated αIibβ3 integrins and displayed increased binding of coagulation factors. Coated platelets, expressing serotonin binding sites, were not identified as a separate population. Thrombin generation and coagulation favored the transformation to phosphatidylserine-exposing platelets with inactivated integrins and reduced adhesion. Prolonged tyrosine phosphorylation in vitro resulted in secondary downregulation of active αIIbβ3. Conclusions These results lead to a new spatial model of thrombus formation, in which aggregated platelets ensure thrombus stability, whereas distinct patches of nonaggregated platelets effectuate procoagulant activity and generate thrombin and fibrin. Herein, the hemostatic activity of a developing thrombus is determined by the balance in formation of proaggregatory and procoagulant platelets. This balance is influenced by antiplatelet and anticoagulant medication. PMID:17761939

  10. Annexin XIIIb Associates with Lipid Microdomains to Function in Apical Delivery

    PubMed Central

    Lafont, Frank; Lecat, Sandra; Verkade, Paul; Simons, Kai

    1998-01-01

    A member of the annexin XIII sub-family, annexin XIIIb, has been implicated in the apical exocytosis of epithelial kidney cells. Annexins are phospholipid-binding proteins that have been suggested to be involved in membrane trafficking events although their actual physiological function remains open. Unlike the other annexins, annexin XIIIs are myristoylated. Here, we show by immunoelectron microscopy that annexin XIIIb is localized to the trans-Golgi network (TGN), vesicular carriers and the apical cell surface. Polarized apical sorting involves clustering of apical proteins into dynamic sphingolipid-cholesterol rafts. We now provide evidence for the raft association of annexin XIIIb. Using in vitro assays and either myristoylated or unmyristoylated recombinant annexin XIIIb, we demonstrate that annexin XIIIb in its native myristoylated form stimulates specifically apical transport whereas the unmyristoylated form inhibits this route. Moreover, we show that formation of apical carriers from the TGN is inhibited by an anti-annexin XIIIb antibody whereas it is stimulated by myristoylated recombinant annexin XIIIb. These results suggest that annexin XIIIb directly participates in apical delivery. PMID:9744874