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Sample records for plasma membrane prevents

  1. Plasma membrane disruption: repair, prevention, adaptation

    Science.gov (United States)

    McNeil, Paul L.; Steinhardt, Richard A.

    2003-01-01

    Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

  2. Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane

    OpenAIRE

    1990-01-01

    The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this c...

  3. Prevention of ligneous conjunctivitis by topical and subconjunctival fresh frozen plasma.

    Science.gov (United States)

    Tabbara, Khalid F

    2004-08-01

    To present a case of ligneous conjunctivitis where the recurrence of membranous conjunctivitis was prevented by subconjunctival and topical instillation of fresh frozen plasma. Interventional case report. A case of ligneous conjunctivitis with multiple recurrences since the age of 3 years developed recurrent membranous conjunctivitis after transconjunctival levator recession. Blood plasminogen activity was determined. The membrane was excised, and the membrane reappeared 4 days later. The patient was treated with excision of the membrane and subconjunctival injection of fresh frozen plasma and topical fresh frozen plasma. Plasminogen activity of the fresh frozen plasma was normal. Plasminogen blood functional activity was 52% (normal is 80%-120%). The patient had complete remission with no recurrences of membranous conjunctivitis after topical and subconjunctival fresh frozen plasma. Prophylactic use of topical and subconjunctival fresh frozen plasma may help in the prevention of membranes in susceptible patients with plasminogen deficiency.

  4. Exploring the biophysical properties of phytosterols in the plasma membrane for novel cancer prevention strategies.

    Science.gov (United States)

    Fakih, Omar; Sanver, Didem; Kane, David; Thorne, James L

    2018-05-03

    Cancer is a global problem with no sign that incidences are reducing. The great costs associated with curing cancer, through developing novel treatments and applying patented therapies, is an increasing burden to developed and developing nations alike. These financial and societal problems will be alleviated by research efforts into prevention, or treatments that utilise off-patent or repurposed agents. Phytosterols are natural components of the diet found in an array of seeds, nuts and vegetables and have been added to several consumer food products for the management of cardio-vascular disease through their ability to lower LDL-cholesterol levels. In this review, we provide a connected view between the fields of structural biophysics and cellular and molecular biology to evaluate the growing evidence that phytosterols impair oncogenic pathways in a range of cancer types. The current state of understanding of how phytosterols alter the biophysical properties of plasma membrane is described, and the potential for phytosterols to be repurposed from cardio-vascular to oncology therapeutics. Through an overview of the types of biophysical and molecular biology experiments that have been performed to date, this review informs the reader of the molecular and biophysical mechanisms through which phytosterols could have anti-cancer properties via their interactions with the plasma cell membrane. We also outline emerging and under-explored areas such as computational modelling, improved biomimetic membranes and ex vivo tissue evaluation. Focus of future research in these areas should improve understanding, not just of phytosterols in cancer cell biology but also to give insights into the interaction between the plasma membrane and the genome. These fields are increasingly providing meaningful biological and clinical data but iterative experiments between molecular biology assays, biosynthetic membrane studies and computational membrane modelling improve and refine our

  5. At the border: the plasma membrane-cell wall continuum.

    Science.gov (United States)

    Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

    2015-03-01

    Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura

    2011-01-01

    The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma me...

  7. Solubilization of rat kidney plasma membrane proteins associated with 3H-aldosterone

    International Nuclear Information System (INIS)

    Ozegovic, B.; Dobrovic-Jenik, D.; Milkovic, S.

    1988-01-01

    The treatment of rat kidney plasma membranes with sodium dodecyl sulphate (SDS) did not essentially affect the ability of the membranes for 3 H-aldosterone binding as compared with the intact plasma membranes (Ozegovic et al., 1977). A gel filtration of 3 H-aldosterone - kidney plasma membranes complex on Sepharose 6B yielded 2 protein and 2 3 H-aldosterone peaks. The proteins which were eluted in the first peak were associated with the first 3 H-aldosterone peak while the second 3 H-aldosterone peak was eluted with Ve corresponding to Ve of free 3 H-aldosterone. Spironolactone, a competitive antagonist of aldosterone, prevented the binding of 3 H-aldosterone to the membrane proteins. The results demonstrated a high affinity of the kidney plasma membranes solubilized with SDS and a specificity of aldosterone binding to the plasma membrane proteins of higher molecular mass. (author)

  8. FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

    Science.gov (United States)

    Lee, Seongju; Chang, Jaerak; Blackstone, Craig

    2016-01-01

    The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

  9. Liver plasma membranes: an effective method to analyze membrane proteome.

    Science.gov (United States)

    Cao, Rui; Liang, Songping

    2012-01-01

    Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

  10. Membrane order in the plasma membrane and endocytic recycling compartment.

    Science.gov (United States)

    Iaea, David B; Maxfield, Frederick R

    2017-01-01

    The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the biophysical properties of membranes. While the properties of model or isolated membranes have been extensively studied, there has been little evaluation of internal membranes in living cells. Here, we use a Nile Red based probe, NR12S, and ratiometric live cell imaging, to analyze the membrane order of the plasma membrane and endocytic recycling compartment. We find that after a brief incubation to allow endocytosis, NR12S is distributed between the plasma membrane and the endocytic recycling compartment. The NR12S reports that the endocytic recycling compartment is more highly ordered than the plasma membrane. We also find that the plasma membrane and the endocytic recycling compartment are differentially affected by altering cellular cholesterol levels. The membrane order of the plasma membrane, but not the endocytic recycling compartment, is altered significantly when cellular cholesterol content is increased or decreased by 20%. These results demonstrate that changes in cellular cholesterol differentially alter membrane order within different organelles.

  11. Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

    Science.gov (United States)

    Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

    2018-01-01

    Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Biogenesis of plasma membrane cholesterol

    International Nuclear Information System (INIS)

    Lange, Y.

    1986-01-01

    A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from [ 3 H] acetate moved to the plasma membrane with a half-time of 1 h at 37 0 C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane

  13. Proteomics and the dynamic plasma membrane

    DEFF Research Database (Denmark)

    Sprenger, Richard R; Jensen, Ole Nørregaard

    2010-01-01

    plasma membrane is of particular interest, by not only serving as a barrier between the "cell interior" and the external environment, but moreover by organizing and clustering essential components to enable dynamic responses to internal and external stimuli. Defining and characterizing the dynamic plasma...... the challenges in functional proteomic studies of the plasma membrane. We review the recent progress in MS-based plasma membrane proteomics by presenting key examples from eukaryotic systems, including mammals, yeast and plants. We highlight the importance of enrichment and quantification technologies required...... for detailed functional and comparative analysis of the dynamic plasma membrane proteome....

  14. Functional implications of plasma membrane condensation for T cell activation.

    Directory of Open Access Journals (Sweden)

    Carles Rentero

    2008-05-01

    Full Text Available The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC, which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

  15. Lipid organization of the plasma membrane

    NARCIS (Netherlands)

    Ingólfsson, Helgi I; Melo, Manuel N; van Eerden, Floris J; Arnarez, Clément; Lopez, Cesar A; Wassenaar, Tsjerk A; Periole, Xavier; de Vries, Alex H; Tieleman, D Peter; Marrink, Siewert J

    2014-01-01

    The detailed organization of cellular membranes remains rather elusive. Based on large-scale molecular dynamics simulations, we provide a high-resolution view of the lipid organization of a plasma membrane at an unprecedented level of complexity. Our plasma membrane model consists of 63 different

  16. The plasma membrane as radiosensitive target

    International Nuclear Information System (INIS)

    Koeteles, Gy.J.

    1986-01-01

    Components and conditions rendering the plasma membrane susceptible for ionizing radiation are discussed. The list of reviews and articles pointing to various aspects of radiation effects on membranes is analyzed. Radiation induced alterations of plasma membrane and energy deposition in cellular microstructures are overviewed. The possible role of membrane alterations in the fate of irradiated cell is also discussed. (author)

  17. Rupturing Giant Plasma Membrane Vesicles to Form Micron-sized Supported Cell Plasma Membranes with Native Transmembrane Proteins.

    Science.gov (United States)

    Chiang, Po-Chieh; Tanady, Kevin; Huang, Ling-Ting; Chao, Ling

    2017-11-09

    Being able to directly obtain micron-sized cell blebs, giant plasma membrane vesicles (GPMVs), with native membrane proteins and deposit them on a planar support to form supported plasma membranes could allow the membrane proteins to be studied by various surface analytical tools in native-like bilayer environments. However, GPMVs do not easily rupture on conventional supports because of their high protein and cholesterol contents. Here, we demonstrate the possibility of using compression generated by the air-water interface to efficiently rupture GPMVs to form micron-sized supported membranes with native plasma membrane proteins. We demonstrated that not only lipid but also a native transmembrane protein in HeLa cells, Aquaporin 3 (AQP3), is mobile in the supported membrane platform. This convenient method for generating micron-sized supported membrane patches with mobile native transmembrane proteins could not only facilitate the study of membrane proteins by surface analytical tools, but could also enable us to use native membrane proteins for bio-sensing applications.

  18. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    Science.gov (United States)

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  19. The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans.

    Science.gov (United States)

    Alvarez, Francisco J; Douglas, Lois M; Rosebrock, Adam; Konopka, James B

    2008-12-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Delta mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Delta mutant are similar to the effects of inhibiting beta-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.

  20. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    Science.gov (United States)

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains. PMID:18799621

  1. Plasma membrane isolation using immobilized concanavalin A magnetic beads.

    Science.gov (United States)

    Lee, Yu-Chen; Srajer Gajdosik, Martina; Josic, Djuro; Lin, Sue-Hwa

    2012-01-01

    Isolation of highly purified plasma membranes is the key step in constructing the plasma membrane proteome. Traditional plasma membrane isolation method takes advantage of the differential density of organelles. While differential centrifugation methods are sufficient to enrich for plasma membranes, the procedure is lengthy and results in low recovery of the membrane fraction. Importantly, there is significant contamination of the plasma membranes with other organelles. The traditional agarose affinity matrix is suitable for isolating proteins but has limitation in separating organelles due to the density of agarose. Immobilization of affinity ligands to magnetic beads allows separation of affinity matrix from organelles through magnets and could be developed for the isolation of organelles. We have developed a simple method for isolating plasma membranes using lectin concanavalin A (ConA) magnetic beads. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. The ConA magnetic beads are used to bind glycosylated proteins present in the membranes. The bound membranes are solubilized from the magnetic beads with a detergent containing the competing sugar alpha methyl mannoside. In this study, we describe the procedure of isolating rat liver plasma membranes using sucrose density gradient centrifugation as described by Neville. We then further purify the membrane fraction by using ConA magnetic beads. After this purification step, main liver plasma membrane proteins, especially the highly glycosylated ones and proteins containing transmembrane domains could be identified by LC-ESI-MS/MS. While not described here, the magnetic bead method can also be used to isolate plasma membranes from cell lysates. This membrane purification method should expedite the cataloging of plasma membrane proteome.

  2. Constitutive activation of a plasma membrane H+-ATPase prevents abscisic acid-mediated stomatal closure

    Science.gov (United States)

    Merlot, Sylvain; Leonhardt, Nathalie; Fenzi, Francesca; Valon, Christiane; Costa, Miguel; Piette, Laurie; Vavasseur, Alain; Genty, Bernard; Boivin, Karine; Müller, Axel; Giraudat, Jérôme; Leung, Jeffrey

    2007-01-01

    Light activates proton (H+)-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO2 to photosynthetic tissues. Light to darkness transition, high CO2 levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H+-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO2 and darkness. The OST2 gene encodes the major plasma membrane H+-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H+-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure. PMID:17557075

  3. Membrane raft association is a determinant of plasma membrane localization.

    Science.gov (United States)

    Diaz-Rohrer, Blanca B; Levental, Kandice R; Simons, Kai; Levental, Ilya

    2014-06-10

    The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

  4. Hydrogen superpermeable membrane operation under plasma conditions

    International Nuclear Information System (INIS)

    Bacal, M.; Bruneteau, A.M.; Livshits, A.I.; Alimov, V.N.; Notkin, M.E.

    2003-01-01

    The effect of ion bombardment on hydrogen plasma-driven permeation through a superpermeable niobium membrane was investigated. It was found that the increase of membrane temperature and the doping of membrane material with oxygen results in the decrease of ion bombardment effect and in permeability increase. It was demonstrated that membrane decarbonization leads to the formation of a membrane state resistant to sputtering. Possible applications of the membrane resistant to ion bombardment as plasma facing components are considered

  5. Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

    OpenAIRE

    Doudová, Lenka

    2017-01-01

    Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

  6. Caveolae as plasma membrane sensors, protectors and organizers.

    Science.gov (United States)

    Parton, Robert G; del Pozo, Miguel A

    2013-02-01

    Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

  7. Autophagosomal membranes assemble at ER-plasma membrane contact sites.

    Science.gov (United States)

    Nascimbeni, Anna Chiara; Codogno, Patrice; Morel, Etienne

    2017-01-01

    The biogenesis of autophagosome, the double membrane bound organelle related to macro-autophagy, is a complex event requiring numerous key-proteins and membrane remodeling events. Our recent findings identify the extended synaptotagmins, crucial tethers of Endoplasmic Reticulum-plasma membrane contact sites, as key-regulators of this molecular sequence.

  8. Experimental study of membrane pump for plasma devices

    International Nuclear Information System (INIS)

    Suzuki, Hajime; Ohyabu, Nobuyoshi; Nakamura, Yukio; Sagara, Akio; Motojima, Osamu; Livshits, A.; Notkin, M.; Busnyuk, A.; Komatsu, Kazuyuki

    1998-01-01

    Recycling control is a key to improve fusion plasma performance. The membrane pump has potential advantages for hydrogen pumping in fusion devices. However, there are unsolved issues for using membrane pump in LHD (Large Helical Device). The first issue is characteristics of the membrane pump under high incident hydrogen atom flux. The second issue is relationship between the surface condition and the pumping efficiency. Impurities from plasma may change the surface condition of the membrane. In order to solve these issues, a membrane pump system was fabricated and installed in a linear plasma device at NIFS (National Institute for Fusion Science). The membrane pump was successfully operated. (author)

  9. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    International Nuclear Information System (INIS)

    Haylett, T.; Thilo, L.

    1986-01-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

  10. Membrane fusion by VAMP3 and plasma membrane t-SNAREs

    International Nuclear Information System (INIS)

    Hu Chuan; Hardee, Deborah; Minnear, Fred

    2007-01-01

    Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

  11. The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

    OpenAIRE

    Alvarez, Francisco J.; Douglas, Lois M.; Rosebrock, Adam; Konopka, James B.

    2008-01-01

    The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenes...

  12. The plasma membrane proteome of germinating barley embryos

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Jensen, O.N.

    2009-01-01

    Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined...... with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two-phase partitioning. Reversed-phase chromatography on C-4 resin performed in micro-spin columns with stepwise elution by 2-propanol...... was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty-one proteins in 14 SDS-PAGE bands were identified by LC-MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination....

  13. Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

    Science.gov (United States)

    Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

    2015-01-01

    It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs.

    Science.gov (United States)

    Tetteroo, P A; Bluemink, J G; Dictus, W J; van Zoelen, E J; de Laat, S W

    1984-07-01

    The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein ("HEDAF") and 5-(N-tetradecanoyl)aminofluorescein ("TEDAF") as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method (D much less than 10(-10) cm2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 X 10(-8) cm2/sec; TEDAF, D = 2.4 X 10(-8) cm2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

  15. Deposition of polymeric perfluored thin films in proton ionic membranes by plasma processes

    International Nuclear Information System (INIS)

    Polak, Peter Lubomir; Mousinho, Ana Paula; Ordonez, Nelson; Silva Zambom, Luis da; Mansano, Ronaldo Domingues

    2007-01-01

    In this work the surfaces of polymeric membranes based on Nafion (proton conducting material), used in proton exchange membranes fuel cells (PEMFC) had been modified by plasma deposition of perfluored polymers, in order to improve its functioning in systems of energy generation (fuel cells). The deposition increases the chemical resistance of the proton ionic polymers without losing the electrical properties. The processing of the membranes also reduces the permeability of the membranes to the alcohols (methanol and ethanol), thus preventing poisoning of the fuel cell. The processing of the membranes of Nafion was carried through in a system of plasma deposition using a mixture of CF 4 and H 2 gases. The plasma processing was made mainly to increase the chemical resistance and result in hydrophobic surfaces. The Fourier transformed infrared (FTIR) technique supplies a spectrum with information about the CF n bond formation. Through the Rutherford back scattering (RBS) technique it was possible to verify the deposition rate of the polymeric layer. The plasma process with composition of 60% of CF 4 and 40% of H 2 presented the best deposition rate. By the spectrum analysis for the optimized configuration, it was possible to verify that the film deposition occurred with a thickness of 90 nm, and fluorine concentration was nearly 30%. Voltammetry made possible to verify that the fluorination increases the membranes chemical resistance, improving the stability of Nafion, becoming an attractive process for construction of fuel cells

  16. Isolation of plasma membrane-associated membranes from rat liver.

    Science.gov (United States)

    Suski, Jan M; Lebiedzinska, Magdalena; Wojtala, Aleksandra; Duszynski, Jerzy; Giorgi, Carlotta; Pinton, Paolo; Wieckowski, Mariusz R

    2014-02-01

    Dynamic interplay between intracellular organelles requires a particular functional apposition of membrane structures. The organelles involved come into close contact, but do not fuse, thereby giving rise to notable microdomains; these microdomains allow rapid communication between the organelles. Plasma membrane-associated membranes (PAMs), which are microdomains of the plasma membrane (PM) interacting with the endoplasmic reticulum (ER) and mitochondria, are dynamic structures that mediate transport of proteins, lipids, ions and metabolites. These structures have gained much interest lately owing to their roles in many crucial cellular processes. Here we provide an optimized protocol for the isolation of PAM, PM and ER fractions from rat liver that is based on a series of differential centrifugations, followed by the fractionation of crude PM on a discontinuous sucrose gradient. The procedure requires ∼8-10 h, and it can be easily modified and adapted to other tissues and cell types.

  17. Plant plasma membrane proteomics for improving cold tolerance

    Directory of Open Access Journals (Sweden)

    Daisuke eTakahashi

    2013-04-01

    Full Text Available Plants are always exposed to various stresses. We have focused on freezing stress, which causes serious problems for agricultural management. When plants suffer freeze-induced damage, the plasma membrane is thought to be the primary site of injury because of its central role in regulation of various cellular processes. Cold tolerant species, however, adapt to such freezing conditions by modifying cellular components and functions (cold acclimation. One of the most important adaptation mechanisms to freezing is alteration of plasma membrane compositions and functions. Advanced proteomic technologies have succeeded in identification of many candidates that may play roles in adaptation of the plasma membrane to freezing stress. Proteomics results suggest that adaptations of plasma membrane functions to low temperature are associated with alterations of protein compositions during cold acclimation. Some of proteins identified by proteomic approaches have been verified their functional roles in freezing tolerance mechanisms further. Thus, accumulation of proteomic results in the plasma membrane is of importance for application to molecular breeding efforts to increase cold tolerance in crops.

  18. Light-induced modification of plant plasma membrane ion transport.

    Science.gov (United States)

    Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

    2010-09-01

    Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

  19. Plasma membrane wounding and repair in pulmonary diseases.

    Science.gov (United States)

    Cong, Xiaofei; Hubmayr, Rolf D; Li, Changgong; Zhao, Xiaoli

    2017-03-01

    Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures, and gastric acid aspiration stress lung cells, and the compromise of plasma membranes occurs as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. Because it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes and discuss relevant genetic, physical, and biological injury mechanisms, as well as mechanisms used by lung cells for cell survival and membrane repair. Finally, we discuss relevant signaling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signaling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells, should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases. Copyright © 2017 the American Physiological Society.

  20. Bile acids modulate signaling by functional perturbation of plasma membrane domains.

    Science.gov (United States)

    Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

    2013-12-13

    Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

  1. Nanoclustering as a dominant feature of plasma membrane organization

    NARCIS (Netherlands)

    Garcia-Parajo, M.F.; Cambi, A.; Torreno-Pina, J.A.; Thompson, N.; Jacobson, K.

    2014-01-01

    Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at

  2. Annexins are instrumental for efficient plasma membrane repair in cancer cells.

    Science.gov (United States)

    Lauritzen, Stine Prehn; Boye, Theresa Louise; Nylandsted, Jesper

    2015-09-01

    Plasma membrane stress can cause damage to the plasma membrane, both when imposed by the extracellular environment and by enhanced oxidative stress. Cells cope with these injuries by rapidly activating their plasma membrane repair system, which is triggered by Ca(2+) influx at the wound site. The repair system is highly dynamic, depends on both lipid and protein components, and include cytoskeletal reorganization, membrane replacements, and membrane fusion events. Cancer cells experience enhanced membrane stress when navigating through dense extracellular matrix, which increases the frequency of membrane injuries. In addition, increased motility and oxidative stress further increase the risk of plasma membrane lesions. Cancer cells compensate by overexpressing Annexin proteins including Annexin A2 (ANXA2). Annexin family members can facilitate membrane fusion events and wound healing by binding to negatively charged phospholipids in the plasma membrane. Plasma membrane repair in cancer cells depends on ANXA2 protein, which is recruited to the wound site and forms a complex with the Ca(2+)-binding EF-hand protein S100A11. Here they regulate actin accumulation around the wound perimeter, which is required for wound closure. In this review, we will discuss the requirement for Annexins, S100 proteins and actin cytoskeleton in the plasma membrane repair response of cancer cells, which reveals a novel avenue for targeting metastatic cancers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Plasma-polymerized alkaline anion-exchange membrane: Synthesis and structure characterization

    International Nuclear Information System (INIS)

    Hu Jue; Meng Yuedong; Zhang Chengxu; Fang Shidong

    2011-01-01

    After-glow discharge plasma polymerization was developed for alkaline anion-exchange membranes synthesis using vinylbenzyl chloride as monomer. X-ray photoelectron spectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were used to characterize the chemical structure properties of plasma-polymerized membranes. Ion-exchange capacities of quaternized poly(vinylbenzyl chloride) (QPVBC) membranes were measured to evaluate their capability of hydroxyl ion transport. A mechanism of plasma polymerization using VBC as monomer that accounts for the competitive effects of free radicals polymerization and plasma ablation in the plasma polymerization process was proposed. Our results indicate that plasma discharge power influences the contents of functional groups and the structure of the plasma polymer membranes, which attribute to the coactions of polymerization and ablation. The properties of uniform morphology, good adhesion to the substrate, high thermal stability and satisfying anion conduction level suggest the potential application of QPVBC membrane deposited at discharge power of 20 W in alkaline direct methanol fuel cells.

  4. Surface modification of nanoporous alumina membranes by plasma polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Losic, Dusan; Cole, Martin A; Dollmann, Bjoern; Vasilev, Krasimir; Griesser, Hans J [Ian Wark Research Institute, University of South Australia, Mawson Lakes, Adelaide, SA 5095 (Australia)], E-mail: dusan.losic@unisa.edu.au

    2008-06-18

    The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200 nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes.

  5. Surface modification of nanoporous alumina membranes by plasma polymerization

    International Nuclear Information System (INIS)

    Losic, Dusan; Cole, Martin A; Dollmann, Bjoern; Vasilev, Krasimir; Griesser, Hans J

    2008-01-01

    The deposition of plasma polymer coatings onto porous alumina (PA) membranes was investigated with the aim of adjusting the surface chemistry and the pore size of the membranes. PA membranes from commercial sources with a range of pore diameters (20, 100 and 200 nm) were used and modified by plasma polymerization using n-heptylamine (HA) monomer, which resulted in a chemically reactive polymer surface with amino groups. Heptylamine plasma polymer (HAPP) layers with a thickness less than the pore diameter do not span the pores but reduce their diameter. Accordingly, by adjusting the deposition time and thus the thickness of the plasma polymer coating, it is feasible to produce any desired pore diameter. The structural and chemical properties of modified membranes were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM) and x-ray electron spectroscopy (XPS). The resultant PA membranes with specific surface chemistry and controlled pore size are applicable for molecular separation, cell culture, bioreactors, biosensing, drug delivery, and engineering complex composite membranes

  6. Giant plasma membrane vesicles: models for understanding membrane organization.

    Science.gov (United States)

    Levental, Kandice R; Levental, Ilya

    2015-01-01

    The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Characteristics of polyimide-based composite membranes fabricated by low-temperature plasma polymerization

    International Nuclear Information System (INIS)

    Dung Thi Tran; Mori, Shinsuke; Suzuki, Masaaki

    2008-01-01

    Composite membranes were prepared by the deposition of plasma-polymerized allylamine films onto a porous polyimide substrate. The relationship between the plasma conditions and the membrane characteristics was described in terms of monomer flow rate, plasma discharge power, plasma polymerization time, and so on. Scanning electron microscope (SEM) images indicate that the thickness of the plasma polymer layer increased and the membrane skin pore size decreased gradually with the increasing of plasma polymerization time. Fourier transform infrared (FTIR) spectra demonstrate the appearance of amine groups in the plasma deposited polymer and the contact angle measurements indicate that the hydrophilicity of the membrane surfaces increased significantly after plasma polymerization. The composite membranes can reject salt from sodium chloride feed solution, and membrane separation performance depends strongly on the plasma conditions applied during the preparation of the plasma deposited polymer films

  8. Plasma modified PLA electrospun membranes for actinorhodin production intensification in Streptomyces coelicolor immobilized-cell cultivations.

    Science.gov (United States)

    Scaffaro, Roberto; Lopresti, Francesco; Sutera, Alberto; Botta, Luigi; Fontana, Rosa Maria; Gallo, Giuseppe

    2017-09-01

    Most of industrially relevant bioproducts are produced by submerged cultivations of actinomycetes. The immobilization of these Gram-positive filamentous bacteria on suitable porous supports may prevent mycelial cell-cell aggregation and pellet formation which usually negatively affect actinomycete submerged cultivations, thus, resulting in an improved biosynthetic capability. In this work, electrospun polylactic acid (PLA) membranes, subjected or not to O 2 -plasma treatment (PLA-plasma), were used as support for immobilized-cell submerged cultivations of Streptomyces coelicolor M145. This strain produces different bioactive compounds, including the blue-pigmented actinorhodin (ACT) and red-pigmented undecylprodigiosin (RED), and constitutes a model for the study of antibiotic-producing actinomycetes. Wet contact angles and X-ray photoelectron spectroscopy analysis confirmed the increased wettability of PLA-plasma due to the formation of polar functional groups such as carboxyl and hydroxyl moieties. Scanning electron microscope observations, carried out at different incubation times, revealed that S. coelicolor immobilized-cells created a dense "biofilm-like" mycelial network on both kinds of PLA membranes. Cultures of S. coelicolor immobilized-cells on PLA or PLA-plasma membranes produced higher biomass (between 1.5 and 2 fold) as well as higher levels of RED and ACT than planktonic cultures. In particular, cultures of immobilized-cells on PLA and PLA-plasma produced comparable levels of RED that were approximatively 4 and 5 fold higher than those produced by planktonic cultures, respectively. In contrast, levels of ACT produced by immobilized-cell cultures on PLA and PLA-plasma were different, being 5 and 10 fold higher than those of planktonic cultures, respectively. Therefore, this is study demonstrated the positive influence of PLA membrane on growth and secondary metabolite production in S. coelicolor and also revealed that O 2 -plasma treated PLA membranes

  9. Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane

    Science.gov (United States)

    Chen, Jiangrong; Zhang, Huifeng; Liu, Guochang; Guo, Chungang; Lv, Jinglie; Zhangb, Yushan

    2018-01-01

    Polytetrafluoroethylene (PTFE) is popular membrane material because of its excellent thermal stability, chemical stability and mechanical stability. However, the low surface energy and non-sticky property of PTFE present challenges for modification. In the present study, plasma treatment was performed to improve the surface adhesion of PTFE hollow fiber membrane. The effect of discharge voltage, treatment time on the adhesion of PTFE hollow fiber membrane was symmetrically evaluated. Results showed that the plasma treatment method contributed to improve the surface activity and roughness of PTFE hollow fiber membrane, and the adhesion strength depend significantly on discharge voltage, which was beneficial to seepage pressure of PTFE hollow fiber membrane module. The adhesion strength of PTFE membrane by plasma treated at 220V for 3min reached as high as 86.2 N, far surpassing the adhesion strength 12.7 N of pristine membrane. Furthermore, improvement of content of free radical and composition analysis changes of the plasma modified PTFE membrane were investigated. The seepage pressure of PTFE membrane by plasma treated at 220V for 3min was 0.375 MPa, which means that the plasma treatment is an effective technique to improve the adhesion strength of membrane.

  10. Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

    Science.gov (United States)

    Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

    2009-12-01

    A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

  11. Surface monofunctionalized polymethyl pentene hollow fiber membranes by plasma treatment and hemocompatibility modification for membrane oxygenators

    Science.gov (United States)

    Huang, Xin; Wang, Weiping; Zheng, Zhi; Fan, Wenling; Mao, Chun; Shi, Jialiang; Li, Lei

    2016-01-01

    The hemocompatibility of polymethyl pentene (PMP) hollow fiber membranes (HFMs) was improved through surface modification for membrane oxygenator applications. The modification was performed stepwise with the following: (1) oxygen plasma treatment, (2) functionalization of monosort hydroxyl groups through NaBH4 reduction, and (3) grafting 2-methacryloyloxyethyl phosphorylcholine (MPC) or heparin. SEM, ATR-FTIR, and XPS analyses were conducted to confirm successful grafting during the modification. The hemocompatibility of PMP HFMs was analyzed and compared through protein adsorption, platelet adhesion, and coagulation tests. Pure CO2 and O2 permeation rates, as well as in vitro gas exchange rates, were determined to evaluate the mass transfer properties of PMP HFMs. SEM results showed that different nanofibril topographies were introduced on the HFM surface. ATR-FTIR and XPS spectra indicated the presence of functionalization of monosort hydroxyl group and the grafting of MPC and heparin. Hemocompatibility evaluation results showed that the modified PMP HFMs presented optimal hemocompatibility compared with pristine HFMs. Gas permeation results revealed that gas permeation flux increased in the modified HFMs because of dense surface etching during the plasma treatment. The results of in vitro gas exchange rates showed that all modified PMP HFMs presented decreased gas exchange rates because of potential surface fluid wetting. The proposed strategy exhibits a potential for fabricating membrane oxygenators for biomedical applications to prevent coagulation formation and alter plasma-induced surface topology and composition.

  12. Effect of plasma membrane fluidity on serotonin transport by endothelial cells

    International Nuclear Information System (INIS)

    Block, E.R.; Edwards, D.

    1987-01-01

    To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotinin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of [ 14 C]-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect of fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells

  13. Membrane-based, sedimentation-assisted plasma separator for point-of-care applications.

    Science.gov (United States)

    Liu, Changchun; Mauk, Michael; Gross, Robert; Bushman, Frederic D; Edelstein, Paul H; Collman, Ronald G; Bau, Haim H

    2013-11-05

    Often, high-sensitivity, point-of-care (POC) clinical tests, such as HIV viral load, require large volumes of plasma. Although centrifuges are ubiquitously used in clinical laboratories to separate plasma from whole blood, centrifugation is generally inappropriate for on-site testing. Suitable alternatives are not readily available to separate the relatively large volumes of plasma from milliliters of blood that may be needed to meet stringent limit-of-detection specifications for low-abundance target molecules. We report on a simple-to-use, low-cost, pump-free, membrane-based, sedimentation-assisted plasma separator capable of separating a relatively large volume of plasma from undiluted whole blood within minutes. This plasma separator consists of an asymmetric, porous, polysulfone membrane housed in a disposable chamber. The separation process takes advantage of both gravitational sedimentation of blood cells and size exclusion-based filtration. The plasma separator demonstrated a "blood in-plasma out" capability, consistently extracting 275 ± 33.5 μL of plasma from 1.8 mL of undiluted whole blood within less than 7 min. The device was used to separate plasma laden with HIV viruses from HIV virus-spiked whole blood with recovery efficiencies of 95.5% ± 3.5%, 88.0% ± 9.5%, and 81.5% ± 12.1% for viral loads of 35,000, 3500, and 350 copies/mL, respectively. The separation process is self-terminating to prevent excessive hemolysis. The HIV-laden plasma was then injected into our custom-made microfluidic chip for nucleic acid testing and was successfully subjected to reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP), demonstrating that the plasma is sufficiently pure to support high-efficiency nucleic acid amplification.

  14. An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

    Science.gov (United States)

    Bezrukov, Ludmila; Blank, Paul S; Polozov, Ivan V; Zimmerberg, Joshua

    2009-11-15

    A method to isolate large quantities of directly accessible plasma membrane from attached cells is presented. The method is based on the adhesion of cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water and subsequent washing steps. Optimal conditions for coating glass plates and time for cell attachment were established. No additional chemical or mechanical treatments were used. Contamination of the isolated plasma membrane by cell organelles was less than 5%. The method uses inexpensive, commercially available polylysine and reusable glass plates. Plasma membrane preparations can be made in 15 min. Using this method, we determined that methyl-beta-cyclodextrin differentially extracts cholesterol from fibroblast cells and their plasma membranes and that these differences are temperature dependent. Determination of the cholesterol/phospholipid ratio from intact cells does not reflect methyl-beta-cyclodextrin plasma membrane extraction properties.

  15. Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

    Science.gov (United States)

    Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

    2010-01-01

    The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

  16. A plasma membrane H + ATPase gene is germinationinduced in ...

    African Journals Online (AJOL)

    A plasma membrane H + ATPase gene is germinationinduced in wheat embryos. ... African Journal of Biotechnology ... of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods.

  17. Composite plasma polymerized sulfonated polystyrene membrane for PEMFC

    Energy Technology Data Exchange (ETDEWEB)

    Nath, Bhabesh Kumar; Khan, Aziz; Chutia, Joyanti, E-mail: jchutiaiasst@gmail.com

    2015-10-15

    Highlights: • Methyl methane sulfonate (MMS) is used as the sulfonating agent. • The proton conductivity of the membrane is found to be 0.141 S cm{sup −1}. • Power density of fuel cell with styrene/MMS membrane is 0.5 W cm{sup −2}. • The membrane exhibits thermal stability up to 140 °C. - Abstract: This work presents the introduction of an organic compound methyl methane sulfonate (MMS) for the first time in fabrication of polystyrene based proton exchange membrane (PEM) by plasma polymerization process. The membrane is fabricated by co-polymerizing styrene and MMS in capacitively coupled continuous RF plasma. The chemical composition of the plasma polymerized polymer membrane is investigated using Fourier Transform Infrared Spectroscopy which reveals the formation of composite structure of styrene and MMS. The surface morphology studied using AFM and SEM depicts the effect of higher partial pressure of MMS on surface topography of the membrane. The proton transport property of the membrane studied using electrochemical impedance spectroscopy shows the achievement of maximum proton conductivity of 0.141 S cm{sup −1} which is comparable to Nafion 117 membrane. Fuel cell performance test of the synthesized membrane shows a maximum power density of 500 mW cm{sup −2} and current density of 0.62 A cm{sup −2} at 0.6 V.

  18. Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

    Science.gov (United States)

    Kraft, Mary L

    2016-01-01

    Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

  19. Changes in plasma membrane structure upon irradiation on thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1993-01-01

    Thymocytes were irradiated with doses of 4 to 10 4 Gy. The binding of 1-anilinonaphtalene-8-sulphonate and Ca 2+ to plasma membranes; viscosity and lipid peroxidation; Stern-Folmer constant; and the number of Sh-groups of membrane proteins were determined. The structural changes in plasma membranes after irradiation of thymocytes were found to be cooperative

  20. Protein diffusion in plant cell plasma membranes: the cell-wall corral.

    Science.gov (United States)

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  1. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    International Nuclear Information System (INIS)

    Witt, P.L.; Bownds, M.D.

    1987-01-01

    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

  2. Solute removal capacity of high cut-off membrane plasma separators.

    Science.gov (United States)

    Ohkubo, Atsushi; Kurashima, Naoki; Nakamura, Ayako; Miyamoto, Satoko; Iimori, Soichiro; Rai, Tatemitsu

    2013-10-01

    In vitro blood filtration was performed by a closed circuit using high cut-off membrane plasma separators, EVACURE EC-2A10 (EC-2A) and EVACURE EC-4A10 (EC-4A). Samples were obtained from sampling sites before the plasma separator, after each plasma separator, and from the ultrafiltrate of each separator. The sieving coefficient (S.C.) of total protein (TP), albumin (Alb), IgG, interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), fibrinogen (Fib), antithrombin III (AT-III), and coagulation factor XIII (FXIII) were calculated. The S.C. of each solute using EC-2A and EC-A4 were as follows; TP: 0.25 and 0.56, Alb: 0.32 and 0.73, IgG: 0.16 and 0.50, IL-6:0.73 and 0.95, IL-8:0.85 and 0.82, TNF-α: 1.07 and 0.99, Fib: 0 and 0, FXIII: 0.07 and 0.17, respectively. When compared with the conventional type of membrane plasma separators, EVACURE could efficiently remove cytokines while retaining coagulation factors such as fibrinogen. Moreover, EC-2A prevented protein loss, whereas EC-4A could remove approximately 50% of IgG. © 2013 The Authors. Therapeutic Apheresis and Dialysis © 2013 International Society for Apheresis.

  3. Protein diffusion in plant cell plasma membranes: The cell-wall corral

    Directory of Open Access Journals (Sweden)

    Alexandre eMartinière

    2013-12-01

    Full Text Available Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment.

  4. Plasma membrane and salinity tolerance of barley plants

    International Nuclear Information System (INIS)

    Al-Rahmani, F. H.; Al-Mashhadani, M. S.; Al-Delemee, N. H.

    1997-01-01

    Barley cultivar, California Mario ut, was grown in a nutrient solution containing increasing Nacl concentrations up to 250 mm. The effect of Nacl on growth, mineral compost ion ant integrity of the plasma membrane was studied. Growth of the shoot'and root was stimulated or little affected by 10 and 20 ml Nacl. Further increase in Nacl concentrations depressed the growth. The depression was conspicuous between 100 and 250 mm Nacl. Increasing Nacl concentration decreased potassium content in the shoots and roots and led to steep increase in sodium accumulation. The integrity of the plasma membrane was measured in term of potassium leakage from the root tips. Rapid leakage of potassium was obtained at Nacl concentrations ranging from 100 to 250 mm. At the same concentrations of Nacl, adenosine triphosphatase activity in the root tips was increased. Results indicate that the plasma membrane of root cells was damaged by the increased levels of salinity. It was concluded that the plasma membrane of root cells is the primary site of salinity toxicity. (authors). 40 refs., 5 tabs. 3 figs

  5. Quantitative changes in adipocyte plasma membrane in response to nutritional manipulations

    International Nuclear Information System (INIS)

    Lewis, D.S.; Masoro, E.J.; Yu, B.P.

    1981-01-01

    The effects of changes in adipocyte size and the effects of nutritional manipulations on the quantity of plasma membrane per adipocyte were investigated. A method for estimating the quantity of plasma membrane was developed based on the specific labeling of adipocyte plasma membrane protein with the nonpermeable labeling agent 125I-labeled diazotized diiodosulfanilic acid. By studying rats (ranging in age from 50 to 125 days) fed a standard laboratory chow or a low fat diet or a high fat diet, a wide range of mean fat cell sizes was obtained. It was found that as the volume of the fat cell increased, the amount of plasma membrane increased in a linear fashion and that this linear relationship had the same slope whether the size of the adipocyte increased slowly with age or rapidly in response to a high fat diet. In contrast, fasting for up to 3 days caused a marked decrease in the mean volume of the adipocytes, but either no change or much less change in the amount of plasma membrane per cell than would have been predicted from the linear relationship between adipocytes, but either no change or much less change in the amount of plasma membrane per cell than would have been predicted form the linear relationship between adipocyte volume and amount of plasma membrane per cell obtained with fed rats, i.e., adipocytes from fasted rats contain more plasma membrane per cell than do fat cells of the same size from fed rats. Neither feeding a high fat diet nor fasting caused detectable changes in the protein and lipid composition of the adipocyte plasma membrane

  6. Protein-centric N-glycoproteomics analysis of membrane and plasma membrane proteins.

    Science.gov (United States)

    Sun, Bingyun; Hood, Leroy

    2014-06-06

    The advent of proteomics technology has transformed our understanding of biological membranes. The challenges for studying membrane proteins have inspired the development of many analytical and bioanalytical tools, and the techniques of glycoproteomics have emerged as an effective means to enrich and characterize membrane and plasma-membrane proteomes. This Review summarizes the development of various glycoproteomics techniques to overcome the hurdles formed by the unique structures and behaviors of membrane proteins with a focus on N-glycoproteomics. Example contributions of N-glycoproteomics to the understanding of membrane biology are provided, and the areas that require future technical breakthroughs are discussed.

  7. Isolation and characterization of the plasma membrane from the yeast Pichia pastoris.

    Science.gov (United States)

    Grillitsch, Karlheinz; Tarazona, Pablo; Klug, Lisa; Wriessnegger, Tamara; Zellnig, Günther; Leitner, Erich; Feussner, Ivo; Daum, Günther

    2014-07-01

    Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Isolation and characterization of plasma membranes from guinea pig ileum

    International Nuclear Information System (INIS)

    Anon.

    1986-01-01

    A plasma membrane fraction from guinea pig ileum has been isolated by extraction of a crude microsomal fraction with a low ionic strength buffer containing ATP and Ca 2+ . The extracted microsomes were subjected to sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membranes were substantially free from contamination with contractile proteins, mitochondria and sarco-plasmic reticulum. The plasma membrane vesicles were enriched 30-to-40-fold in Na + -K + -ATPase and 5'-nucleotidase activities. The plasma membrane vesicles accumulated Ca 2+ in the presence of ATP. The addition of Ca 2+ ionophore A23187 to vesicles loaded with Ca 2+ in the presence of ATP removed Ca 2+ completely from the vesicles in one minute. The Km values for the Ca 2+ -dependent phosphorylated intermediates of Ca 2+ -Mg 2+ -ATPase and Ca 2+ uptake were approximately 0.8 μM indicating that the phosphorylated intermediates represent phosphorylation of Ca 2+ pump ATPase. The 3 H-nitrendipine binding to plasma membranes was characterized by high affinity with Kd of 185 pM and B/sub max/ 1280 fmol/mg protein. The plasma membrane vesicles prepared by these procedures can prove useful for the study of ion transport

  9. Cholesterol asymmetry in synaptic plasma membranes.

    Science.gov (United States)

    Wood, W Gibson; Igbavboa, Urule; Müller, Walter E; Eckert, Gunter P

    2011-03-01

    Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  10. Mechanisms underlying anomalous diffusion in the plasma membrane.

    Science.gov (United States)

    Krapf, Diego

    2015-01-01

    The plasma membrane is a complex fluid where lipids and proteins undergo diffusive motion critical to biochemical reactions. Through quantitative imaging analyses such as single-particle tracking, it is observed that diffusion in the cell membrane is usually anomalous in the sense that the mean squared displacement is not linear with time. This chapter describes the different models that are employed to describe anomalous diffusion, paying special attention to the experimental evidence that supports these models in the plasma membrane. We review models based on anticorrelated displacements, such as fractional Brownian motion and obstructed diffusion, and nonstationary models such as continuous time random walks. We also emphasize evidence for the formation of distinct compartments that transiently form on the cell surface. Finally, we overview heterogeneous diffusion processes in the plasma membrane, which have recently attracted considerable interest. Copyright © 2015. Published by Elsevier Inc.

  11. Properties of Plasma Membrane from Pea Root Seedlings under Altered Gravity

    Science.gov (United States)

    Klymchuk, D.; Baranenko, V.; Vorobyova, T. V.; Kurylenko, I.; Chyzhykova, O.; Dubovoy, V.

    In this study, the properties of pea (Pisum sativum L.) plasma membrane were examined to determine how the membrane structure and functions are regulated in response to clinorotation (2 rev/min) conditions. Membrane preparations enriched by plasma membrane vesicles were obtained by aqueous two-phase partitioning from 6-day seedling roots. The specific characteristics of H^+-ATPase, lípid composition and peroxidation intensity as well as fluidity of lipid bilayer were analysed. ATP hydrolytic activity was inhibited by ortovanadate and was insensitive to aside and nitrate in sealed plasma membrane vesicles isolated from both clinorotated and control seedlings. Plasma membrane vesicles from clinorotated seedlings in comparison to controls were characterised by increase in the total lipid/protein ratio, ATP hydrolytic activity and intensifying of lipid peroxidation. Sitosterol and campesterol were the predominant free sterol species. Clinorotated seedlings contained a slightly higher level of unsaturated fatty acid than controls. Plasma membrane vesicles were labelled with pyrene and fluorescence originating from monomeric (I_M) molecules and excimeric (I_E) aggregates were measured. The calculated I_E/I_M values were higher in clinorotated seedlings compared with controls reflecting the reduction in membrane microviscosity. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H^+-ATPase activity in response of pea seedlings to altered gravity is discussed.

  12. Plasma deposited fluorinated films on porous membranes

    Energy Technology Data Exchange (ETDEWEB)

    Gancarz, Irena [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Bryjak, Marek, E-mail: marek.bryjak@pwr.edu.pl [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Kujawski, Jan; Wolska, Joanna [Department of Polymer and Carbon Materials, Wrocław University of Technology, 50-370 Wrocław (Poland); Kujawa, Joanna; Kujawski, Wojciech [Nicolaus Copernicus University, Faculty of Chemistry, 7 Gagarina St., 87-100 Torun (Poland)

    2015-02-01

    75 KHz plasma was used to modify track etched poly(ethylene terephthalate) membranes and deposit on them flouropolymers. Two fluorine bearing monomers were used: perflourohexane and hexafluorobenzene. The modified surfaces were analyzed by means of attenuated total reflection infra-red spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, atomic force microscopy and wettability. It was detected that hexaflourobenxene deposited to the larger extent than perflourohaxane did. The roughness of surfaces decreased when more fluoropolymer was deposited. The hydrophobic character of surface slightly disappeared during 20-days storage of hexaflourobenzene modified membrane. Perfluorohexane modified membrane did not change its character within 120 days after modification. It was expected that this phenomenon resulted from post-reactions of oxygen with radicals in polymer deposits. The obtained membranes could be used for membrane distillation of juices. - Highlights: • Plasma deposited hydrophobic layer of flouropolymers. • Deposition degree affects the surface properties. • Hydrohilization of surface due to reaction of oxygen with entrapped radicals. • Possibility to use modified porous membrane for water distillation and apple juice concentration.

  13. Plants and fungi in the era of heterogeneous plasma membranes.

    Science.gov (United States)

    Opekarová, M; Malinsky, J; Tanner, W

    2010-09-01

    Examples from yeast and plant cells are described that show that their plasma membrane is laterally compartmented. Distinct lateral domains encompassing both specific lipids and integral proteins coexist within the plane of the plasma membrane. The compartments are either spatially stable and include distinct sets of proteins, or they are transiently formed to accomplish diverse functions. They are not related to lipid rafts or their clusters, as defined for mammalian cells. This review summarises only well-documented compartments of plasma membranes from plants and fungi, which have been recognised using microscopic approaches. In several cases, physiological functions of the membrane compartmentation are revealed.

  14. Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane with highly effective blood compatibility via atmospheric plasma-induced surface copolymerization.

    Science.gov (United States)

    Chang, Yung; Chang, Wan-Ju; Shih, Yu-Ju; Wei, Ta-Chin; Hsiue, Ging-Ho

    2011-04-01

    Development of nonfouling membranes to prevent nonspecific protein adsorption and platelet adhesion is critical for many biomedical applications. It is always a challenge to control the surface graft copolymerization of a highly polar monomer from the highly hydrophobic surface of a fluoropolymer membrane. In this work, the blood compatibility of poly(vinylidene fluoride) (PVDF) membranes with surface-grafted electrically neutral zwitterionic poly(sulfobetaine methacrylate) (PSBMA), from atmospheric plasma-induced surface copolymerization, was studied. The effect of surface composition and graft morphology, electrical neutrality, hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Blood compatibility of the zwitterionic PVDF membranes was systematically evaluated by plasma protein adsorption, platelet adhesion, plasma-clotting time, and blood cell hemolysis. It was found that the nonfouling nature and hydration capability of grafted PSBMA polymers can be effectively controlled by regulating the grafting coverage and charge balance of the PSBMA layer on the PVDF membrane surface. Even a slight charge bias in the grafted zwitterionic PSBMA layer can induce electrostatic interactions between proteins and the membrane surfaces, leading to surface protein adsorption, platelet activation, plasma clotting and blood cell hemolysis. Thus, the optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities when comes into contact with human blood. © 2011 American Chemical Society

  15. Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

    International Nuclear Information System (INIS)

    Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

    1987-01-01

    The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

  16. Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

    Science.gov (United States)

    Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

    2017-06-14

    Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

  17. The dynamics of plant plasma membrane proteins: PINs and beyond.

    Science.gov (United States)

    Luschnig, Christian; Vert, Grégory

    2014-08-01

    Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment. © 2014. Published by The Company of Biologists Ltd.

  18. Atmospheric-pressure plasma activation and surface characterization on polyethylene membrane separator

    Science.gov (United States)

    Tseng, Yu-Chien; Li, Hsiao-Ling; Huang, Chun

    2017-01-01

    The surface hydrophilic activation of a polyethylene membrane separator was achieved using an atmospheric-pressure plasma jet. The surface of the atmospheric-pressure-plasma-treated membrane separator was found to be highly hydrophilic realized by adjusting the plasma power input. The variations in membrane separator chemical structure were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Chemical analysis showed newly formed carbonyl-containing groups and high surface concentrations of oxygen-containing species on the atmospheric-pressure-plasma-treated polymeric separator surface. It also showed that surface hydrophilicity primarily increased from the polar component after atmospheric-pressure plasma treatment. The surface and pore structures of the polyethylene membrane separator were examined by scanning electron microscopy, revealing a slight alteration in the pore structure. As a result of the incorporation of polar functionalities by atmospheric-pressure plasma activation, the electrolyte uptake and electrochemical impedance of the atmospheric-pressure-plasma-treated membrane separator improved. The investigational results show that the separator surface can be controlled by atmospheric-pressure plasma surface treatment to tailor the hydrophilicity and enhance the electrochemical performance of lithium ion batteries.

  19. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+ -ATPase by preventing interaction with 14-3-3 protein

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Guo, Yan; Cuin, Tracey A.

    2007-01-01

    Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report...... that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, is a negative regulator of the plasma membrane proton pump (PM Hþ-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phosphorylates the PM Hþ-ATPase AHA2 at a novel...

  20. Estradiol-mediated internalisation of the non-activated estrogen receptor from the goat uterine plasma membrane: identification of the proteins involved.

    Science.gov (United States)

    Sreeja, S; Thampan, Raghava Varman

    2004-04-01

    An indirect approach has been made to study the molecular details associated with the estradiol-induced internalisation of the non-activated estrogen receptor (naER) from the goat uterine plasma membrane. The internalisation of naER appears to be an energy dependent process. Exposure of the plasma membrane to estradiol results in the activation of a Mg2+ dependent ATPase associated with the membrane fraction. Presence of quercetin in the medium prevented the activation of the Mg2+ ATPase as well as the dissociation of naER from the plasma membrane. Using isolated plasma membrane preparations it has been possible to identify the proteins which interact with naER during various stages of its internalisation. The main proteins identified are: (1) a 58 kDa protein, p58, which apparently recognizes the nuclear localization signals on the naER and transports it to the nucleus: (2) hsp70: (3) hsp90, the functional roles of which remain unknown at this stage; (4) a 50 kDa protein associated with the clathrin coated vesicles, presumed to be involved in recognizing the tyrosine based internalisation signals on the naER; (5) actin which mediates the plasma membrane-to-nucleus movement of the naER-p58 complex.

  1. Influence of ionizing radiation on the plasma membrane proteins

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1992-01-01

    The effect of ionizing radiation on the meat cattle thymocytes plasma membranes was studied. Using fluorescence quenching technique the effect of irradiation of proteins conformation was investigated. The influence of ionizing radiation on the plasma membranes was shown to be followed by changes of the protein structure-dynamic organization

  2. Interaction between La(III) and proteins on the plasma membrane of horseradish

    Science.gov (United States)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  3. RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

    Science.gov (United States)

    Bi, Zhen; Merl-Pham, Juliane; Uehlein, Norbert; Zimmer, Ina; Mühlhans, Stefanie; Aichler, Michaela; Walch, Axel Karl; Kaldenhoff, Ralf; Palme, Klaus; Schnitzler, Jörg-Peter; Block, Katja

    2015-10-14

    Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement. Copyright © 2015. Published by Elsevier B.V.

  4. Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.

    Science.gov (United States)

    Lund, Anette; Fuglsang, Anja Thoe

    2012-01-01

    Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.

  5. Layered plasma polymer composite membranes

    Science.gov (United States)

    Babcock, Walter C.

    1994-01-01

    Layered plasma polymer composite fluid separation membranes are disclosed, which comprise alternating selective and permeable layers for a total of at least 2n layers, where n is .gtoreq.2 and is the number of selective layers.

  6. Long-Time Plasma Membrane Imaging Based on a Two-Step Synergistic Cell Surface Modification Strategy.

    Science.gov (United States)

    Jia, Hao-Ran; Wang, Hong-Yin; Yu, Zhi-Wu; Chen, Zhan; Wu, Fu-Gen

    2016-03-16

    Long-time stable plasma membrane imaging is difficult due to the fast cellular internalization of fluorescent dyes and the quick detachment of the dyes from the membrane. In this study, we developed a two-step synergistic cell surface modification and labeling strategy to realize long-time plasma membrane imaging. Initially, a multisite plasma membrane anchoring reagent, glycol chitosan-10% PEG2000 cholesterol-10% biotin (abbreviated as "GC-Chol-Biotin"), was incubated with cells to modify the plasma membranes with biotin groups with the assistance of the membrane anchoring ability of cholesterol moieties. Fluorescein isothiocyanate (FITC)-conjugated avidin was then introduced to achieve the fluorescence-labeled plasma membranes based on the supramolecular recognition between biotin and avidin. This strategy achieved stable plasma membrane imaging for up to 8 h without substantial internalization of the dyes, and avoided the quick fluorescence loss caused by the detachment of dyes from plasma membranes. We have also demonstrated that the imaging performance of our staining strategy far surpassed that of current commercial plasma membrane imaging reagents such as DiD and CellMask. Furthermore, the photodynamic damage of plasma membranes caused by a photosensitizer, Chlorin e6 (Ce6), was tracked in real time for 5 h during continuous laser irradiation. Plasma membrane behaviors including cell shrinkage, membrane blebbing, and plasma membrane vesiculation could be dynamically recorded. Therefore, the imaging strategy developed in this work may provide a novel platform to investigate plasma membrane behaviors over a relatively long time period.

  7. Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

    Science.gov (United States)

    Marette, A; Dimitrakoudis, D; Shi, Q; Rodgers, C D; Klip, A; Vranic, M

    1999-02-01

    We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

  8. Insights into plant plasma membrane aquaporin trafficking.

    Science.gov (United States)

    Hachez, Charles; Besserer, Arnaud; Chevalier, Adrien S; Chaumont, François

    2013-06-01

    Plasma membrane intrinsic proteins (PIPs) are plant aquaporins that facilitate the diffusion of water and small uncharged solutes through the cell membrane. Deciphering the network of interacting proteins that modulate PIP trafficking to and activity in the plasma membrane is essential to improve our knowledge about PIP regulation and function. This review highlights the most recent advances related to PIP subcellular routing and dynamic redistribution, identifies some key molecular interacting proteins, and indicates exciting directions for future research in this field. A better understanding of the mechanisms by which plants optimize water movement might help in identifying new molecular players of agronomical relevance involved in the control of cellular water uptake and drought tolerance. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

    International Nuclear Information System (INIS)

    Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

    1990-01-01

    The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

  10. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

    Science.gov (United States)

    Chen, Z.; Yin, C.; Wang, S.; Ito, K.; Fu, Q. M.; Deng, Q. R.; Fu, P.; Lin, Z. D.; Zhang, Y.

    2017-01-01

    A polysulfone/TiO2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result.

  12. Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

    International Nuclear Information System (INIS)

    Chen, Z; Yin, C; Wang, S; Fu, Q M; Deng, Q R; Fu, P; Lin, Z D; Zhang, Y; Ito, K

    2017-01-01

    A polysulfone/TiO 2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO 2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result. (paper)

  13. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    Science.gov (United States)

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

    Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

  14. Research on Permeability of Poly(ethylene) Terephthalate Track Membranes Modified in Plasma

    CERN Document Server

    Dmitriev, S N; Sleptsov, V V; Elinson, V M; Potrjasaj, V V

    2001-01-01

    The properties of poly(ethylene) terephthalate track membranes subjected to the plasma RF-discharge treatment in air have been investigated. The effect of the treatment conditions in plasma on the structure and the properties of the membranes formed in the gas-discharge etching has been studied. It has been figured out that the influence of the air plasma on the membranes under study leads to a formation of asymmetric membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It is shown that the presence of the modified layer on the surface of the membranes causes changing their hydrodynamic characteristics - water permeability of the membranes treated in plasma in a greater degree depends upon {pH} of the filtered solution.

  15. Dynamics of HIV-1 RNA Near the Plasma Membrane during Virus Assembly.

    Science.gov (United States)

    Sardo, Luca; Hatch, Steven C; Chen, Jianbo; Nikolaitchik, Olga; Burdick, Ryan C; Chen, De; Westlake, Christopher J; Lockett, Stephen; Pathak, Vinay K; Hu, Wei-Shau

    2015-11-01

    To increase our understanding of the events that lead to HIV-1 genome packaging, we examined the dynamics of viral RNA and Gag-RNA interactions near the plasma membrane by using total internal reflection fluorescence microscopy. We labeled HIV-1 RNA with a photoconvertible Eos protein via an RNA-binding protein that recognizes stem-loop sequences engineered into the viral genome. Near-UV light exposure causes an irreversible structural change in Eos and alters its emitted fluorescence from green to red. We studied the dynamics of HIV-1 RNA by photoconverting Eos near the plasma membrane, and we monitored the population of photoconverted red-Eos-labeled RNA signals over time. We found that in the absence of Gag, most of the HIV-1 RNAs stayed near the plasma membrane transiently, for a few minutes. The presence of Gag significantly increased the time that RNAs stayed near the plasma membrane: most of the RNAs were still detected after 30 min. We then quantified the proportion of HIV-1 RNAs near the plasma membrane that were packaged into assembling viral complexes. By tagging Gag with blue fluorescent protein, we observed that only a portion, ∼13 to 34%, of the HIV-1 RNAs that reached the membrane were recruited into assembling particles in an hour, and the frequency of HIV-1 RNA packaging varied with the Gag expression level. Our studies reveal the HIV-1 RNA dynamics on the plasma membrane and the efficiency of RNA recruitment and provide insights into the events leading to the generation of infectious HIV-1 virions. Nascent HIV-1 particles assemble on plasma membranes. During the assembly process, HIV-1 RNA genomes must be encapsidated into viral complexes to generate infectious particles. To gain insights into the RNA packaging and virus assembly mechanisms, we labeled and monitored the HIV-1 RNA signals near the plasma membrane. Our results showed that most of the HIV-1 RNAs stayed near the plasma membrane for only a few minutes in the absence of Gag, whereas

  16. Regulation of the Plasma Membrane H+-ATPase

    DEFF Research Database (Denmark)

    Falhof, Janus

    The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

  17. Hunting for low abundant redox proteins in plant plasma membranes.

    Science.gov (United States)

    Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

    2009-04-13

    Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

  18. Gravity Responsive NADH Oxidase of the Plasma Membrane

    Science.gov (United States)

    Morre, D. James (Inventor)

    2002-01-01

    A method and apparatus for sensing gravity using an NADH oxidase of the plasma membrane which has been found to respond to unit gravity and low centrifugal g forces. The oxidation rate of NADH supplied to the NADH oxidase is measured and translated to represent the relative gravitational force exerted on the protein. The NADH oxidase of the plasma membrane may be obtained from plant or animal sources or may be produced recombinantly.

  19. Preparation of poly(2-chloroaniline) membrane and plasma surface modification

    International Nuclear Information System (INIS)

    Kir, E.; Oksuz, L.; Helhel, S.

    2006-01-01

    P2ClAn membranes were obtained from chemically synthesized poly(2-chloroaniline) (P2ClAn) by casting method. These membranes were cast from dimethyl formamide (DMF) and were in the undoped state. P2ClAn membranes were characterized by Fourier infrared spectroscopy and scanning electron microscopy. Measurements of water content capacity, membrane thickness and ion-exchange capacity of the cast membranes were carried out. P2ClAn membranes were treated by electron cylotron resonance (ECR) plasma for surface modification. Plasma treatment has been successfully utilized for improving the surface properties of P2ClAn membranes such as increasing pore diameters and number of pores for better anion or molecule transportation

  20. Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

    OpenAIRE

    Kelly, K L; Kiechle, F L; Jarett, L

    1984-01-01

    Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of...

  1. Membrane oscillations in the channel of a stationary plasma motor

    International Nuclear Information System (INIS)

    Bugrova, A.I.; Lipatov, A.S.; Morozov, A.I.; Kharchevnikov, V.K.

    1999-01-01

    Results of measuring the ion flux density in the channel of the stationary plasma drive are presented. Two plane easters move both along and transverse to the plasma flux. During the experiment, the strong low-frequency oscillations (∼ 35 kHz) are observed in the channel of the stationary plasma drive. It is found that membrane oscillations are accompanied by oscillations of the electron temperature. These membrane oscillations affect the divergence of the output plasma jet and the erosion of the output part of the channel of the stationary plasma drive [ru

  2. Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

    Science.gov (United States)

    Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

    2017-05-01

    The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Research on permeability of poly(ethylene) terephthalate track membranes modified in plasma

    International Nuclear Information System (INIS)

    Dmitriev, S.N.; Kravets, L.I.; Sleptsov, V.V.; Elinson, V.M.; Potryasaj, V.V.

    2001-01-01

    The properties of poly(ethylene) terephthalate track membranes subjected to the plasma RF-discharge treatment in air have been investigated. The effect of the treatment conditions in plasma on the structure and the properties of the membranes formed in the gas-discharge etching has been studied. It has been figured out that the influence of the air plasma on the membranes under study leads to a formation of asymmetric membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It is shown that the presence of the modified layer on the surface of the membranes causes changing their hydrodynamic characteristics - water permeability of the membranes treated in plasma in a greater degree depends upon pH of the filtered solution. (author)

  4. Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

    International Nuclear Information System (INIS)

    Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

    1986-01-01

    Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

  5. Exclusive photorelease of signalling lipids at the plasma membrane.

    Science.gov (United States)

    Nadler, André; Yushchenko, Dmytro A; Müller, Rainer; Stein, Frank; Feng, Suihan; Mulle, Christophe; Carta, Mario; Schultz, Carsten

    2015-12-21

    Photoactivation of caged biomolecules has become a powerful approach to study cellular signalling events. Here we report a method for anchoring and uncaging biomolecules exclusively at the outer leaflet of the plasma membrane by employing a photocleavable, sulfonated coumarin derivative. The novel caging group allows quantifying the reaction progress and efficiency of uncaging reactions in a live-cell microscopy setup, thereby greatly improving the control of uncaging experiments. We synthesized arachidonic acid derivatives bearing the new negatively charged or a neutral, membrane-permeant coumarin caging group to locally induce signalling either at the plasma membrane or on internal membranes in β-cells and brain slices derived from C57B1/6 mice. Uncaging at the plasma membrane triggers a strong enhancement of calcium oscillations in β-cells and a pronounced potentiation of synaptic transmission while uncaging inside cells blocks calcium oscillations in β-cells and causes a more transient effect on neuronal transmission, respectively. The precise subcellular site of arachidonic acid release is therefore crucial for signalling outcome in two independent systems.

  6. Novel determinants of H-Ras plasma membrane localization and transformation

    DEFF Research Database (Denmark)

    Willumsen, B M; Cox, A D; Solski, P A

    1996-01-01

    cysteine did not abolish palmitoylation. However, despite continued lipid modification the mutant proteins failed to bind to plasma membranes and instead accumulated on internal membranes and, importantly, were not transforming. Addition of an N-terminal myristoylation signal to these defective mutants......, or to proteins entirely lacking the C-terminal 25 residues restored both plasma membrane association and transforming activity. Thus, H-Ras does not absolutely require prenylation or palmitoylation nor indeed its hypervariable domain in order to interact with effectors that ultimately cause transformation....... However, in this native state, the C-terminus appears to provide a combination of lipids and a previously unrecognized signal for specific plasma membrane targeting that are essential for the correct localization and biological function of H-Ras....

  7. Na+/H+ exchange activity in the plasma membrane of Arabidopsis.

    Science.gov (United States)

    Qiu, Quan-Sheng; Barkla, Bronwyn J; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S

    2003-06-01

    In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.

  8. Reverse-osmosis membranes by plasma polymerization

    Science.gov (United States)

    Hollahan, J. R.; Wydeven, T.

    1972-01-01

    Thin allyl amine polymer films were developed using plasma polymerization. Resulting dry composite membranes effectively reject sodium chloride during reverse osmosis. Films are 98% sodium chloride rejective, and 46% urea rejective.

  9. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1991-01-01

    This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

  10. Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

    Science.gov (United States)

    Luo, Yu; Russinova, Eugenia

    2017-01-01

    Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

  11. Plasma membrane proteomics and its application in clinical cancer biomarker discovery

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Lund, Rikke; Ditzel, Henrik J

    2010-01-01

    Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions...... targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate...... cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal...

  12. A highly phosphorylated subpopulation of insulin-like growth factor II/mannose 6-phosphate receptors is concentrated in a clathrin-enriched plasma membrane fraction

    International Nuclear Information System (INIS)

    Corvera, S.; Folander, K.; Clairmont, K.B.; Czech, M.P.

    1988-01-01

    Insulin-like growth factor II (IGF-II)/mannose 6-phosphate (Man-6-P) receptors immunoprecipitated from purified plasma membranes of 32 P-labeled rat adipocytes are markedly heterogenous in their phosphorylation state. Approximately 80% of the plasma membrane receptors are solubilized in 1% (vol/vol) Triton X-100 and are phosphorylated on serine residues at a stoichiometry of ∼ 0.1-0.2 mol of phosphate per mol of receptor. In contrast, 15-20% of the receptors are Triton X-100-insoluble and are phosphorylated on serine and threonine residues at ∼ 4 or 5 mol of phosphate per mol of receptor. This Triton X-100-insoluble membrane subfraction contains only 5% of the total plasma membrane protein and yet contains all of the clathrin heavy chain associated with plasma membrane. Based on the relative yields of protein in the detergent-insoluble material, IGF-II/Man-6-P receptors are concentrated ∼ 3-fold in this clathrin-enriched subfraction. Taken together, these results indicate that insulin decreases the phosphorylation state of a highly phosphorylated subpopulation of IGF-II/Man-6-P receptors on the plasma membrane. In addition, insulin action may prevent the concentration of these receptors in a clathrin-enriched membrane subfraction

  13. Plasma membrane aquaporins mediates vesicle stability in broccoli.

    Directory of Open Access Journals (Sweden)

    Maria Del Carmen Martínez-Ballesta

    Full Text Available The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.

  14. Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus.

    Science.gov (United States)

    Adu-Gyamfi, Emmanuel; Johnson, Kristen A; Fraser, Mark E; Scott, Jordan L; Soni, Smita P; Jones, Keaton R; Digman, Michelle A; Gratton, Enrico; Tessier, Charles R; Stahelin, Robert V

    2015-09-01

    Lipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles. The lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. Copyright © 2015, American

  15. Plasma surface modification of polypropylene track-etched membrane to improve its performance properties

    Science.gov (United States)

    Kravets, L. I.; Elinson, V. M.; Ibragimov, R. G.; Mitu, B.; Dinescu, G.

    2018-02-01

    The surface and electrochemical properties of polypropylene track-etched membrane treated by plasma of nitrogen, air and oxygen are studied. The effect of the plasma-forming gas composition on the surface morphology is considered. It has been found that the micro-relief of the membrane surface formed under the gas-discharge etching, changes. Moreover, the effect of the non-polymerizing gas plasma leads to formation of oxygen-containing functional groups, mostly carbonyl and carboxyl. It is shown that due to the formation of polar groups on the surface and its higher roughness, the wettability of the plasma-modified membranes improves. In addition, the presence of polar groups on the membrane surface layer modifies its electrochemical properties so that conductivity of plasma-treated membranes increase.

  16. Effect of Plasma Membrane Semipermeability in Making the Membrane Electric Double Layer Capacitances Significant.

    Science.gov (United States)

    Sinha, Shayandev; Sachar, Harnoor Singh; Das, Siddhartha

    2018-01-30

    Electric double layers (or EDLs) formed at the membrane-electrolyte interface (MEI) and membrane-cytosol interface (MCI) of a charged lipid bilayer plasma membrane develop finitely large capacitances. However, these EDL capacitances are often much larger than the intrinsic capacitance of the membrane, and all of these capacitances are in series. Consequently, the effect of these EDL capacitances in dictating the overall membrane-EDL effective capacitance C eff becomes negligible. In this paper, we challenge this conventional notion pertaining to the membrane-EDL capacitances. We demonstrate that, on the basis of the system parameters, the EDL capacitance for both the permeable and semipermeable membranes can be small enough to influence C eff . For the semipermeable membranes, however, this lowering of the EDL capacitance can be much larger, ensuring a reduction of C eff by more than 20-25%. Furthermore, for the semipermeable membranes, the reduction in C eff is witnessed over a much larger range of system parameters. We attribute such an occurrence to the highly nonintuitive electrostatic potential distribution associated with the recently discovered phenomena of charge-inversion-like electrostatics and the attainment of a positive zeta potential at the MCI for charged semipermeable membranes. We anticipate that our findings will impact the quantification and the identification of a large number of biophysical phenomena that are probed by measuring the plasma membrane capacitance.

  17. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    Science.gov (United States)

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins. Copyright © 2016 Elsevier GmbH. All rights reserved.

  18. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

  19. Plasma treatment of polyethersulfone membrane for benzene removal from water by air gap membrane distillation.

    Science.gov (United States)

    Pedram, Sara; Mortaheb, Hamid Reza; Arefi-Khonsari, Farzaneh

    2018-01-01

    In order to obtain a durable cost-effective membrane for membrane distillation (MD) process, flat sheet polyethersulfone (PES) membranes were modified by an atmospheric pressure nonequilibrium plasma generated using a dielectric barrier discharge in a mixture of argon and hexamethyldisiloxane as the organosilicon precursor. The surface properties of the plasma-modified membranes were characterized by water contact angle (CA), liquid entry pressure, X-ray photoelectron spectroscopy, scanning electron microscopy, and atomic force microscopy. The water CA of the membrane was increased from 64° to 104° by depositing a Si(CH 3 )-rich thin layer. While the pristine PES membrane was not applicable in the MD process, the modified PES membrane could be applied for the first time in an air gap membrane distillation setup for the removal of benzene as a volatile organic compound from water. The experimental design using central composite design and response surface methodology was applied to study the effects of feed temperature, concentration, and flow rate as well as their binary interactions on the overall permeate flux and separation factor. The separation factor and permeation flux of the modified PES membrane at optimum conditions were comparable with those of commercial polytetrafluoroethylene membrane.

  20. Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

    International Nuclear Information System (INIS)

    Cook, H.W.; Palmer, F.B.; Byers, D.M.; Spence, M.W.

    1988-01-01

    A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl- 3 H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane

  1. Research on water permeability of poly(ethylene) terephthalate track membranes modified with plasma

    International Nuclear Information System (INIS)

    Kravets, L.I.; Dmitriev, S.N.; Sleptsov, V.V.; Elinson, V.M.; Potryasay, V.V.

    2001-01-01

    The properties of poly(ethylene) terephthalate track membranes subjected to effect of plasma of the RF-discharge in air have been investigated. The influence conditions of a plasma treatment on the surface properties and hydrodynamic characteristics of the membranes has been studied. It has been found that the effect of the air plasma on the researched membranes results in a formation of asymmetric track membranes with a higher flow rate, the structure and chemical composition of their superficial layer are changed. It was shown that the availability of the modified layer on the membrane surface caused changing in their hydrodynamic characteristics - the water permeability of the membranes, processed in plasma, in a greater degree depends upon pH of a filtered solution. (author)

  2. TEMPERATURE DEPENDENT PHASE BEHAVIOR AND PROTEIN PARTITIONING IN GIANT PLASMA MEMBRANE VESICLES

    OpenAIRE

    Johnson, SA; Stinson, BM; Go, M; Carmona, LM; Reminick, JI; Fang, X; Baumgart, T

    2010-01-01

    Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured...

  3. G-protein activity in Percoll-purified plasma membranes, bulk plasma membranes, and low-density plasma membranes isolated from rat cerebral cortex

    Czech Academy of Sciences Publication Activity Database

    Bouřová, Lenka; Stöhr, Jiří; Lisý, Václav; Rudajev, Vladimír; Novotný, Jiří; Svoboda, Petr

    2009-01-01

    Roč. 15, č. 4 (2009), BR111-BR122 ISSN 1234-1010 R&D Projects: GA MŠk(CZ) LC554; GA MŠk(CZ) LC06063; GA ČR(CZ) GA309/06/0121; GA AV ČR(CZ) IAA500110606 Institutional research plan: CEZ:AV0Z50110509 Keywords : rat cerebral cortex * plasma membrane * G-protein activity Subject RIV: CE - Biochemistry Impact factor: 1.543, year: 2009

  4. Towards Enhanced Performance Thin-film Composite Membranes via Surface Plasma Modification

    Science.gov (United States)

    Reis, Rackel; Dumée, Ludovic F.; Tardy, Blaise L.; Dagastine, Raymond; Orbell, John D.; Schutz, Jürg A.; Duke, Mikel C.

    2016-01-01

    Advancing the design of thin-film composite membrane surfaces is one of the most promising pathways to deal with treating varying water qualities and increase their long-term stability and permeability. Although plasma technologies have been explored for surface modification of bulk micro and ultrafiltration membrane materials, the modification of thin film composite membranes is yet to be systematically investigated. Here, the performance of commercial thin-film composite desalination membranes has been significantly enhanced by rapid and facile, low pressure, argon plasma activation. Pressure driven water desalination tests showed that at low power density, flux was improved by 22% without compromising salt rejection. Various plasma durations and excitation powers have been systematically evaluated to assess the impact of plasma glow reactions on the physico-chemical properties of these materials associated with permeability. With increasing power density, plasma treatment enhanced the hydrophilicity of the surfaces, where water contact angles decreasing by 70% were strongly correlated with increased negative charge and smooth uniform surface morphology. These results highlight a versatile chemical modification technique for post-treatment of commercial membrane products that provides uniform morphology and chemically altered surface properties. PMID:27363670

  5. Plasma Membranes Modified by Plasma Treatment or Deposition as Solid Electrolytes for Potential Application in Solid Alkaline Fuel Cells

    Science.gov (United States)

    Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

    2012-01-01

    In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane. PMID:24958295

  6. Plasma membranes modified by plasma treatment or deposition as solid electrolytes for potential application in solid alkaline fuel cells.

    Science.gov (United States)

    Reinholdt, Marc; Ilie, Alina; Roualdès, Stéphanie; Frugier, Jérémy; Schieda, Mauricio; Coutanceau, Christophe; Martemianov, Serguei; Flaud, Valérie; Beche, Eric; Durand, Jean

    2012-07-30

    In the highly competitive market of fuel cells, solid alkaline fuel cells using liquid fuel (such as cheap, non-toxic and non-valorized glycerol) and not requiring noble metal as catalyst seem quite promising. One of the main hurdles for emergence of such a technology is the development of a hydroxide-conducting membrane characterized by both high conductivity and low fuel permeability. Plasma treatments can enable to positively tune the main fuel cell membrane requirements. In this work, commercial ADP-Morgane® fluorinated polymer membranes and a new brand of cross-linked poly(aryl-ether) polymer membranes, named AMELI-32®, both containing quaternary ammonium functionalities, have been modified by argon plasma treatment or triallylamine-based plasma deposit. Under the concomitant etching/cross-linking/oxidation effects inherent to the plasma modification, transport properties (ionic exchange capacity, water uptake, ionic conductivity and fuel retention) of membranes have been improved. Consequently, using plasma modified ADP-Morgane® membrane as electrolyte in a solid alkaline fuel cell operating with glycerol as fuel has allowed increasing the maximum power density by a factor 3 when compared to the untreated membrane.

  7. Plasma membrane of a marine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins

    International Nuclear Information System (INIS)

    Crumpton, M.J.; Marchalonis, J.J.; Haustein, D.; Atwell, J.L.; Harris, A.W.

    1976-01-01

    Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125 I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase, T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125 I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells. (author)

  8. Severe Hemolysis in a Patient With Erythrocytosis During Coupled Plasma Filtration Adsorption Therapy Was Prevented by Changing From Membrane-Based Technique to a Centrifuge-Based One.

    Science.gov (United States)

    Fan, Rong; Wu, Buyun; Kong, Ling; Gong, Dehua

    2016-01-01

    Coupled plasma filtration adsorption (CPFA) usually adopts membrane to separate plasma from blood. Here, we reported a case with erythrocytosis experienced severe hemolysis and membrane rupture during CPFA, which was avoided by changing from membrane-based technique to a centrifuge-based one. A 66-year-old man was to receive CPFA for severe hyperbilirubinemia (total bilirubin 922 μmol/L, direct bilirubin 638 μmol/L) caused by obstruction of biliary tract. He had erythrocytosis (hemoglobin 230 g/L, hematocrit 0.634) for years because of untreated tetralogy of Fallot. Severe hemolysis and membrane rupture occurred immediately after blood entering into the plasma separator even at a low flow rate (50 mL/min) and persisted after changing a new separator. Finally, centrifugal plasma separation technique was used for CPFA in this patient, and no hemolysis occurred. After 3 sessions of CPFA, total bilirubin level decreased to 199 μmol/L with an average decline by 35% per session. Thereafter, the patient received endoscopic biliary stent implantation, and total bilirubin level returned to nearly normal. Therefore, centrifugal-based plasma separation can also be used in CPFA and may be superior to a membrane-based one in patients with hyperviscosity.

  9. There Is No Simple Model of the Plasma Membrane Organization

    Science.gov (United States)

    Bernardino de la Serna, Jorge; Schütz, Gerhard J.; Eggeling, Christian; Cebecauer, Marek

    2016-01-01

    Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure. PMID:27747212

  10. Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

    Science.gov (United States)

    Takahashi, Daisuke; Li, Bin; Nakayama, Takato; Kawamura, Yukio; Uemura, Matsuo

    2014-01-01

    Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

  11. Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

    OpenAIRE

    Scheifele, Lisa Z.; Rhoads, Jonathan D.; Parent, Leslie J.

    2003-01-01

    Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affin...

  12. Plasma-modified polyethylene membrane as a separator for lithium-ion polymer battery

    International Nuclear Information System (INIS)

    Kim, Jun Young; Lee, Yongbeom; Lim, Dae Young

    2009-01-01

    The surface of polyethylene (PE) membranes as a separator for lithium-ion polymer battery was modified with acrylonitrile (AN) using the plasma technology. The plasma-induced acrylonitrile coated PE (PiAN-PE) membrane was characterized by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and contact angle measurement. The electrochemical performance of the lithium-ion polymer cell fabricated with the PE and the PiAN-PE membranes were also analyzed. The surface characterization demonstrates that the enhanced adhesion of the PiAN-PE membrane resulted from the increased polar component of surface energy for the PiAN-PE membrane. The presence of the PiAN induced onto the surface of the membrane via the plasma modification plays a critical role in improving the wettability and electrolyte retention, the interfacial adhesion between the electrodes and the separator, the cycle performance of the resulting lithium-ion polymer cell assembly. The PiAN-PE membrane modified by the plasma treatment holds a great potential to be used as a high-performance and cost-effective separator for lithium-ion polymer battery.

  13. Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

    Science.gov (United States)

    Sych, Taras; Mély, Yves; Römer, Winfried

    2018-05-26

    The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

  14. Plasma membrane organization and dynamics is probe and cell line dependent.

    Science.gov (United States)

    Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

    2017-09-01

    The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue

  15. Tissue Factor Coagulant Activity is Regulated by the Plasma Membrane Microenvironment.

    Science.gov (United States)

    Yu, Yuanjie; Böing, Anita N; Hau, Chi M; Hajji, Najat; Ruf, Wolfram; Sturk, Auguste; Nieuwland, Rienk

    2018-06-01

     Tissue factor (TF) can be present in a non-coagulant and coagulant form. Whether the coagulant activity is affected by the plasma membrane microenvironment is unexplored.  This article studies the presence and coagulant activity of human TF in plasma membrane micro-domains.  Plasma membranes were isolated from human MIA PaCa2 cells, MDA-MB-231 cells and human vascular smooth muscle cells by Percoll gradient ultracentrifugation after cell disruption. Plasma membranes were fractionated by OptiPrep gradient ultracentrifugation, and the presence of TF, flotillin, caveolin, clathrin, protein disulphide isomerase (PDI), TF pathway inhibitor (TFPI) and phosphatidylserine (PS) were determined.  Plasma membranes contain two detergent-resistant membrane (DRM) compartments differing in density and biochemical composition. High-density DRMs (DRM-H) have a density ( ρ ) of 1.15 to 1.20 g/mL and contain clathrin, whereas low-density DRMs (DRM-L) have a density between 1.09 and 1.13 g/mL and do not contain clathrin. Both DRMs contain TF, flotillin and caveolin. PDI is detectable in DRM-H, TFPI is not detectable in either DMR-H or DRM-L and PS is detectable in DRM-L. The DRM-H-associated TF (> 95% of the TF antigen) lacks detectable coagulant activity, whereas the DRM-L-associated TF triggers coagulation. This coagulant activity is inhibited by lactadherin and thus PS-dependent, but seemed insensitive to 16F16, an inhibitor of PDI.  Non-coagulant and coagulant TF are present within different types of DRMs in the plasma membrane, and the composition of these DRMs may affect the TF coagulant activity. Schattauer GmbH Stuttgart.

  16. Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

    Science.gov (United States)

    Heyno, Eiri; Mary, Véronique; Schopfer, Peter; Krieger-Liszkay, Anja

    2011-07-01

    Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

  17. Photostable bipolar fluorescent probe for video tracking plasma membranes related cellular processes.

    Science.gov (United States)

    Zhang, Xinfu; Wang, Chao; Jin, Liji; Han, Zhuo; Xiao, Yi

    2014-08-13

    Plasma membranes can sense the stimulations and transmit the signals from extracellular environment and then make further responses through changes in locations, shapes or morphologies. Common fluorescent membrane markers are not well suited for long time tracking due to their shorter retention time inside plasma membranes and/or their lower photostability. To this end, we develop a new bipolar marker, Mem-SQAC, which can stably insert into plasma membranes of different cells and exhibits a long retention time over 30 min. Mem-SQAC also inherits excellent photostability from the BODIPY dye family. Large two-photon absorption cross sections and long wavelength fluorescence emissions further enhance the competitiveness of Mem-SQAC as a membrane marker. By using Mem-SQAC, significant morphological changes of plasma membranes have been monitored during heavy metal poisoning and drug induced apoptosis of MCF-7 cells; the change tendencies are so distinctly different from each other that they can be used as indicators to distinguish different cell injuries. Further on, the complete processes of endocytosis toward Staphylococcus aureus and Escherichia coli by RAW 264.7 cells have been dynamically tracked. It is discovered that plasma membranes take quite different actions in response to the two bacteria, information unavailable in previous research reports.

  18. ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

    Science.gov (United States)

    Isono, Erika; Kalinowska, Kamila

    2017-12-01

    To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Fluidity of pea root plasma membranes under altered gravity

    Science.gov (United States)

    Klymchuk, D. O.; Baranenko, V. V.; Vorobyova, T. V.; Dubovoy, V. D.

    This investigation aims to determine whether clinorotation 2 rev min of pea Pisum sativum L seedlings induces the alterations in the physical-chemical properties of cellular membranes including the plasma membrane fluidity The last is an important regulator of functional activity of membrane enzymes The plasma membranes were isolated by aqueous two-phase partitioning from roots of 6-day old pea seedlings The membrane fluidity was examined by fluorescence spectroscopy using pyrene probe The plasma membrane vesicles with known protein concentration were added to the incubation buffer to a final concentration of 50 mu g of protein per ml A small amount by 1 mu l of pyrene solution in 2-propanol was added to the incubation mixture to a final probe concentration 5 mu M at constant mixing Fluorescence spectra were measured using a Perkin-Elmer LS-50 spectrofluorometer Perkin-Elmer England Pyrene was excited at 337 nm and fluorescence intensity of monomers I M and excimers I E were measured at 393 and 470 nm respectively The I E I M ratios were 0 081 pm 0 003 and 0 072 pm 0 004 in preparations obtained from clinorotated and the control seedlings respectively This fact indicates that rotation on the clinostat increases the membrane fluidity Compared with controls clinorotated seedlings have also showed a reduced growth and a higher level of total unsaturated fatty acids determined by gas chromatography The factors that influence on the fluidity of membrane lipids in bilayer appear to be the

  20. Androgen Receptor Localizes to Plasma Membrane by Binding to Caveolin-1 in Mouse Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Qiong Deng

    2017-01-01

    Full Text Available The nonclassical androgen signaling pathway translates signals into alterations in cellular function within minutes, and this action is proposed to be mediated by an androgen receptor (AR localized to the plasma membrane. This study was designed to determine the mechanism underlying the membrane association of androgen receptor in TM4 cells, a mouse Sertoli cell line. Western blot analysis indicated testosterone-induced AR translocation to the cell membrane. Data from coimmunoprecipitation indicated that AR is associated with caveolin-1, and testosterone enhanced this association. Knockdown of caveolin-1 by shRNA decreased the amount of AR localized to membrane fraction and prevented AR membrane trafficking after being exposed to testosterone at physiological concentration. The palmitoylation inhibitor 2-bromopalmitate decreased AR membrane localization in basal condition and completely blocked testosterone-induced AR translocation to membrane fraction. These data suggested that AR localized to membrane fraction by binding with caveolin-1 through palmitoylation of the cysteine residue. This study provided a new evidence for AR membrane localization and its application for clarifying the nonclassical signaling pathway of androgens.

  1. Role of phosphatidylinositol 4,5-bisphosphate in regulating EHD2 plasma membrane localization.

    Directory of Open Access Journals (Sweden)

    Laura C Simone

    Full Text Available The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4 play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2 plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy, and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

  2. Surface modification of polyacrylonitrile co-polymer membranes using pulsed direct current nitrogen plasma

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Dipankar; Neogi, Sudarsan; De, Sirshendu, E-mail: sde@che.iitkgp.ernet.in

    2015-12-31

    Low temperature plasma treatment using pulsed direct current discharge of nitrogen gas was employed to enhance hydrophilicity of the polyacrylonitrile co-polymer membranes. The membranes were characterized in terms of morphology, structure, hydrophilicity, and membrane performance. Properties and functional groups on the surface of polyacrylonitrile co-polymer membranes were investigated by contact angle, scanning electron microscopy, Fourier transform infrared and X-ray photoelectron spectroscopy. Effects of plasma conditions, namely, pulsed voltage, duty cycle and treatment time on increase in membrane hydrophilicity were studied. Permeability of treated membrane was increased by 47% and it was retained up to 70 days. Surface etching due to plasma treatment was confirmed by weight loss of the treated membranes. Due to surface etching, average pore size increased and rejection of 200 kDa polyethylene glycol decreased to about 70% for the treated membrane. Oxygen and nitrogen functional groups were responsible for surface hydrophilicity. - Highlights: • Surface modification of polyacrylonitrile co-polymer membranes by pulsed direct current nitrogen plasma • Hydrophilic functional groups incorporated on the membrane surface • Significant enhancement of the permeability and wettability of the membranes • Water contact angle increased with storage time and finally stabilized.

  3. Surface modification of polyacrylonitrile co-polymer membranes using pulsed direct current nitrogen plasma

    International Nuclear Information System (INIS)

    Pal, Dipankar; Neogi, Sudarsan; De, Sirshendu

    2015-01-01

    Low temperature plasma treatment using pulsed direct current discharge of nitrogen gas was employed to enhance hydrophilicity of the polyacrylonitrile co-polymer membranes. The membranes were characterized in terms of morphology, structure, hydrophilicity, and membrane performance. Properties and functional groups on the surface of polyacrylonitrile co-polymer membranes were investigated by contact angle, scanning electron microscopy, Fourier transform infrared and X-ray photoelectron spectroscopy. Effects of plasma conditions, namely, pulsed voltage, duty cycle and treatment time on increase in membrane hydrophilicity were studied. Permeability of treated membrane was increased by 47% and it was retained up to 70 days. Surface etching due to plasma treatment was confirmed by weight loss of the treated membranes. Due to surface etching, average pore size increased and rejection of 200 kDa polyethylene glycol decreased to about 70% for the treated membrane. Oxygen and nitrogen functional groups were responsible for surface hydrophilicity. - Highlights: • Surface modification of polyacrylonitrile co-polymer membranes by pulsed direct current nitrogen plasma • Hydrophilic functional groups incorporated on the membrane surface • Significant enhancement of the permeability and wettability of the membranes • Water contact angle increased with storage time and finally stabilized.

  4. The Plasma Membrane Calcium Pump

    Science.gov (United States)

    Rasmussen, H.

    1983-01-01

    Three aspect of cellular calcium metabolism in animal cells was discussed including the importance of the plasma membrane in calcium homeostasis, experiments dealing with the actual mechanism of the calcium pump, and the function of the pump in relationship to the mitochondria and to the function of calmodulin in the intact cell.

  5. Function of plasma membrane microdomain-associated proteins during legume nodulation.

    Science.gov (United States)

    Qiao, Zhenzhen; Libault, Marc

    2017-10-03

    Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

  6. Perforin rapidly induces plasma membrane phospholipid flip-flop.

    Directory of Open Access Journals (Sweden)

    Sunil S Metkar

    Full Text Available The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

  7. PLASMA-MEMBRANE LIPID ALTERATIONS INDUCED BY NACL IN WINTER-WHEAT ROOTS

    NARCIS (Netherlands)

    MANSOUR, MMF; VANHASSELT, PR; KUIPER, PJC

    A highly enriched plasma membrane fraction was isolated by two phase partitioning from wheat roots (Triticum aestivum L. cv. Vivant) grown with and without 100 mM NaCl. The lipids of the plasma membrane fraction were extracted and characterized. Phosphatidylcholine and phosphatidylethanolamine were

  8. Active calcium transport in plasma membrane vesicles from developing cotyledons of common bean

    International Nuclear Information System (INIS)

    Huang Jianzhong; Chen Ziyuan

    1995-01-01

    Plasma membrane vesicles were prepared from the developing cotyledons of common bean (Phaseolus vulgaris L cv Diyundou) by aqueous two-phase partitioning and characterized as to their purity by assaying marker enzymes for other membranes. The putative plasma membrane fraction was minimally contaminated by membranes other than plasma membrane and hence was of high purity. It exhibited a Ca 2+ -dependent ATPase activity, which was inhibited by 1 μmol/L EB and promoted by calcium ionophore A23187. Such an activity was responsible for the observed ATP-dependent 45 Ca 2+ uptake into inside-out plasma membrane vesicles. This process was stimulated by 0.6 μmol/L CaM and 20 μmol/L IAA but inhibited by 2 μmol/L ABA and abolished by A23187. Possible role of cytoplasmic Ca 2+ in mediating phytohormones activity is discussed

  9. Zymosterol is located in the plasma membrane of cultured human fibroblasts

    International Nuclear Information System (INIS)

    Echevarria, F.; Norton, R.A.; Nes, W.D.; Lange, Y.

    1990-01-01

    Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. (1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. (2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. (3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool

  10. Na+/H+ Exchange Activity in the Plasma Membrane of Arabidopsis1

    Science.gov (United States)

    Qiu, Quan-Sheng; Barkla, Bronwyn J.; Vera-Estrella, Rosario; Zhu, Jian-Kang; Schumaker, Karen S.

    2003-01-01

    In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt. PMID:12805632

  11. Palmitoylation of POTE family proteins for plasma membrane targeting

    International Nuclear Information System (INIS)

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-01-01

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

  12. Thymocyte plasma membrane of the rainbow trout, Salmo gairdneri: Associated immunoglobulin and heteroantigens

    Science.gov (United States)

    Warr, G.W.; DeLuca, D.; Anderson, D.P.

    1983-01-01

    1. Thymic lymphocytes of the rainbow trout, S. gairdneri were disrupted and a plasma membrane containing fraction isolated by differential and buoyant density centrifugation.2. Radioiodine introduced into the membrane by the lactoperoxidase catalyzed reaction and immunoglobulin (identified by radioimmunoassay with monoclonal antibody) both copurified in the plasma membrane fraction.3. Rabbit antibody raised to the plasma membrane fraction showed a strong reaction with trout lymphocytes in immunofluorescence, was mitogenic for trout lymphocytes, and recognized lymphocyte membrane heteroantigens of molecular weight > 70,000 in the thymus and 45,000–95,000 in the head kidney.

  13. TiO2-Based Phosphoproteomic Analysis of the Plasma Membrane and the Effects of Phosphatase Inhibitor Treatment

    DEFF Research Database (Denmark)

    Thingholm, Tine; Larsen, Martin Røssel; Ingrell, Christian

    2008-01-01

    Phosphorylation of plasma membrane proteins frequently initiates signal transduction pathways or attenuate plasma membrane transport processes. Because of the low abundance and hydrophobic features of many plasma membrane proteins and the low stoichiometry of protein phosphorylation, studies...... of the plasma membrane phosphoproteome are challenging. We present an optimized analytical strategy for plasma membrane phosphoproteomics that combines efficient plasma membrane protein preparation with TiO 2-based phosphopeptide enrichment and high-performance mass spectrometry for phosphopeptide sequencing....... We used sucrose centrifugation in combination with sodium carbonate extraction to achieve efficient and reproducible purification of low microgram levels of plasma membrane proteins from human mesenchymal stem cells (hMSCs, 10 (7) cells), achieving more than 70% yield of membrane proteins...

  14. Fluorescence interference contrast based approach to study real time interaction of melittin with plasma membranes

    Science.gov (United States)

    Gupta, Sharad; Gui, Dong; Zandi, Roya; Gill, Sarjeet; Mohideen, Umar

    2014-03-01

    Melittin is an anti-bacterial and hemolytic toxic peptide found in bee venom. Cell lysis behavior of peptides has been widely investigated, but the exact interaction mechanism of lytic peptides with lipid membranes and its constituents has not been understood completely. In this paper we study the melittin interaction with lipid plasma membranes in real time using non-invasive and non-contact fluorescence interference contrast microscopy (FLIC). Particularly the interaction of melittin with plasma membranes was studied in a controlled molecular environment, where these plasma membrane were composed of saturated lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and unsaturated lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) with and without cholesterol. We found out that melittin starts to form nanometer size pores in the plasma membranes shortly after interacting with membranes. But the addition of cholesterol in plasma membrane slows down the pore formation process. Our results show that inclusion of cholesterol to the plasma membranes make them more resilient towards pore formation and lysis of membrane.

  15. Towards structural and functional analysis of the plant plasma membrane proton pump

    DEFF Research Database (Denmark)

    Justesen, Bo Højen

    The plasma membrane H+-ATPase is a proton pump essential for several physiological important processes in plants. Through the extrusion of protons from the cell, the PM H+-ATPase establishes and maintains a proton gradient used by proton coupled transporters and secondary active transport...... of nutrients and metabolites across the plasma membrane. Additional processes involving the PM H+-ATPase includes plant growth, development, and response to biotic and abiotic stresses. Extensive efforts have been made in attempts to elucidate the detailed physiological role and biochemical characteristics...... of plasma membrane H+-ATPases. Studies on the plasma membrane H+-ATPases have involved both in vivo and in vitro approaches, with the latter employing either solubilisation by detergent micelles, or reconstitution into lipid vesicles. Despite resulting in a large body of information on structure, function...

  16. Transport proteins of the plant plasma membrane

    Science.gov (United States)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  17. Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

    International Nuclear Information System (INIS)

    Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

    1988-01-01

    Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor α subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[γ- 32 P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor β subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins

  18. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  19. A plasma membrane localization signal in the HIV-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into VSV virions.

    Science.gov (United States)

    Johnson, J E; Rodgers, W; Rose, J K

    1998-11-25

    Previous studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated. Copyright 1998 Academic Press.

  20. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

    of plants and fungi to generate electrochemical proton gradients. A recently published crystal structure of a plasma membrane H(+)-ATPase contributes to our knowledge about the mechanism of these essential enzymes. Together with biochemical and structural data presented in this thesis we are now able...

  1. Fibrinogen Reduction During Selective Plasma Exchange due to Membrane Fouling.

    Science.gov (United States)

    Ohkubo, Atsushi; Okado, Tomokazu; Miyamoto, Satoko; Hashimoto, Yurie; Komori, Shigeto; Yamamoto, Motoki; Maeda, Takuma; Itagaki, Ayako; Yamamoto, Hiroko; Seshima, Hiroshi; Kurashima, Naoki; Iimori, Soichiro; Naito, Shotaro; Sohara, Eisei; Uchida, Shinichi; Rai, Tatemitsu

    2017-06-01

    Fibrinogen is substantially reduced by most plasmapheresis modalities but retained in selective plasma exchange using Evacure EC-4A10 (EC-4A). Although EC-4A's fibrinogen sieving coefficient is 0, a session of selective plasma exchange reduced fibrinogen by approximately 19%. Here, we investigated sieving coefficient in five patients. When the mean processed plasma volume was 1.15 × plasma volume, the mean reduction of fibrinogen during selective plasma exchange was approximately 15%. Fibrinogen sieving coefficient was 0 when the processed plasma volume was 1.0 L, increasing to 0.07 when the processed plasma volume was 3.0 L, with a mean of 0.03 during selective plasma exchange. When fibrinogen sieving coefficient was 0, selective plasma exchange reduced fibrinogen by approximately 10%. Scanning electron microscopy images revealed internal fouling of EC-4A's hollow fiber membrane by substances such as fibrinogen fibrils. Thus, fibrinogen reduction by selective plasma exchange may be predominantly caused by membrane fouling rather than filtration. © 2017 International Society for Apheresis, Japanese Society for Apheresis, and Japanese Society for Dialysis Therapy.

  2. Morphology-properties relationship of gas plasma treated hydrophobic meso-porous membranes and their improved performance for desalination by membrane distillation

    International Nuclear Information System (INIS)

    Dumée, Ludovic F.; Alglave, Hortense; Chaffraix, Thomas; Lin, Bao; Magniez, Kevin; Schütz, Jürg

    2016-01-01

    Graphical abstract: - Highlights: • Systematic surface modifications by gas plasma treatment of hydrophobic polymers. • Correlation between plasma parameters and materials surface energy and morphology. • Spectral analysis of the formation of functional groups across the membranes surface. • Relationship between wettability, roughness and performance. - Abstract: The impact on performance of the surface energy and roughness of membrane materials used for direct contact membrane distillation are critical but yet poorly investigated parameters. The capacity to alter the wettability of highly hydrophobic materials such as poly(tetra-fluoro-ethylene) (PTFE) by gas plasma treatments is reported in this paper. An equally important contribution from this investigation arises from illustrating how vaporized material from the treated sample participates after a short while in the composition of the plasma and fundamentally changes the result of surface chemistry processes. The water contact angle across the hydrophobic membranes is generally controlled by varying the plasma gas conditions, such as the plasma power, chamber pressure and irradiation duration. Changes to surface porosity and roughness of the bulk material as well as the surface chemistry, through specific and partial de-fluorination of the surface were detected and systematically studied by Fourier transform infra-red analysis and scanning electron microscopy. It was found that the rupture of fibrils, formed during membrane processing by thermal-stretching, led to the formation of a denser surface composed of nodules similar to these naturally acting as bridging points across the membrane material between fibrils. This structural change has a profound and impart a permanent effect on the permeation across the modified membranes, which was found to be enhanced by up to 10% for long plasma exposures while the selectivity of the membranes was found to remain unaffected by the treatment at a level higher

  3. Morphology-properties relationship of gas plasma treated hydrophobic meso-porous membranes and their improved performance for desalination by membrane distillation

    Energy Technology Data Exchange (ETDEWEB)

    Dumée, Ludovic F., E-mail: ludovic.dumee@deakin.edu.au [Deakin University, Geelong Victoria–Australia - Institute for Frontier Materials (Australia); Alglave, Hortense; Chaffraix, Thomas; Lin, Bao; Magniez, Kevin [Deakin University, Geelong Victoria–Australia - Institute for Frontier Materials (Australia); Schütz, Jürg [CSIRO, Manufacturing Flagship, 75 Pigdons Road, 3216 Waurn Ponds, Victoria (Australia)

    2016-02-15

    Graphical abstract: - Highlights: • Systematic surface modifications by gas plasma treatment of hydrophobic polymers. • Correlation between plasma parameters and materials surface energy and morphology. • Spectral analysis of the formation of functional groups across the membranes surface. • Relationship between wettability, roughness and performance. - Abstract: The impact on performance of the surface energy and roughness of membrane materials used for direct contact membrane distillation are critical but yet poorly investigated parameters. The capacity to alter the wettability of highly hydrophobic materials such as poly(tetra-fluoro-ethylene) (PTFE) by gas plasma treatments is reported in this paper. An equally important contribution from this investigation arises from illustrating how vaporized material from the treated sample participates after a short while in the composition of the plasma and fundamentally changes the result of surface chemistry processes. The water contact angle across the hydrophobic membranes is generally controlled by varying the plasma gas conditions, such as the plasma power, chamber pressure and irradiation duration. Changes to surface porosity and roughness of the bulk material as well as the surface chemistry, through specific and partial de-fluorination of the surface were detected and systematically studied by Fourier transform infra-red analysis and scanning electron microscopy. It was found that the rupture of fibrils, formed during membrane processing by thermal-stretching, led to the formation of a denser surface composed of nodules similar to these naturally acting as bridging points across the membrane material between fibrils. This structural change has a profound and impart a permanent effect on the permeation across the modified membranes, which was found to be enhanced by up to 10% for long plasma exposures while the selectivity of the membranes was found to remain unaffected by the treatment at a level higher

  4. Reorganization of plasma membrane lipid domains during conidial germination.

    Science.gov (United States)

    Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

    2017-02-01

    Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Method for plasma surface treating and preparation of membrane layers

    NARCIS (Netherlands)

    1992-01-01

    The invention relates to an apparatus suitable for plasma surface treating (e.g. forming a membrane layer on a substrate) which comprises a plasma generation section (2) which is in communication via at least one plasma inlet means (4) (e.g. a nozzle) with an enclosed plasma treating section (3)

  6. Plasma deposition of silver nanoparticles on ultrafiltration membranes: antibacterial and anti-biofouling properties.

    Science.gov (United States)

    Cruz, Mercedes Cecilia; Ruano, Gustavo; Wolf, Marcus; Hecker, Dominic; Vidaurre, Elza Castro; Schmittgens, Ralph; Rajal, Verónica Beatriz

    2015-02-01

    A novel and versatile plasma reactor was used to modify Polyethersulphone commercial membranes. The equipment was applied to: i) functionalize the membranes with low-temperature plasmas, ii) deposit a film of poly(methyl methacrylate) (PMMA) by Plasma Enhanced Chemical Vapor Deposition (PECVD) and, iii) deposit silver nanoparticles (SNP) by Gas Flow Sputtering. Each modification process was performed in the same reactor consecutively, without exposure of the membranes to atmospheric air. Scanning electron microscopy and transmission electron microscopy were used to characterize the particles and modified membranes. SNP are evenly distributed on the membrane surface. Particle fixation and transport inside membranes were assessed before- and after-washing assays by X-ray photoelectron spectroscopy depth profiling analysis. PMMA addition improved SNP fixation. Plasma-treated membranes showed higher hydrophilicity. Anti-biofouling activity was successfully achieved against Gram-positive ( Enterococcus faecalis ) and -negative ( Salmonella Typhimurium) bacteria. Therefore, disinfection by ultrafiltration showed substantial resistance to biofouling. The post-synthesis functionalization process developed provides a more efficient fabrication route for anti-biofouling and anti-bacterial membranes used in the water treatment field. To the best of our knowledge, this is the first report of a gas phase condensation process combined with a PECVD procedure in order to deposit SNP on commercial membranes to inhibit biofouling formation.

  7. Polyphosphoinositides are present in plasma membranes isolated from fusogenic carrot cells

    International Nuclear Information System (INIS)

    Wheeler, J.J.; Boss, W.F.

    1987-01-01

    Fusogenic carrot cells grown in suspension culture were labeled 12 hours with myo-[2- 3 H]inositol. Plasma membranes were isolated from the prelabeled fusogenic carrot cells by both aqueous polymer two-phase partitioning and Renografin density gradients. With both methods, the plasma membrane-enriched fractions, as identified by marker enzymes, were enriched in [ 3 H]inositol-labeled phosphatidylinositol monophosphate (PIP) and phosphatidylinositol bisphosphate (PIP 2 ). An additional [ 3 H]inositol-labeled lipid, lysophosphatidylinositol monophosphate, which migrated between PIP and PIP 2 on thin layer plates, was found primarily in the plasma membrane-rich fraction of the fusogenic cells. This was in contrast to lysophosphatidylinositol which is found primarily in the lower phase, microsomal/mitchrondrial-rich fraction

  8. A Loss in the Plasma Membrane ATPase Activity and Its Recovery Coincides with Incipient Freeze-Thaw Injury and Postthaw Recovery in Onion Bulb Scale Tissue 1

    Science.gov (United States)

    Arora, Rajeev; Palta, Jiwan P.

    1991-01-01

    Plasma membrane ATPase has been proposed to be functionally altered during early stages of injury caused by a freeze-thaw stress. Complete recovery from freezing injury in onion cells during the postthaw period provided evidence in support of this proposal. During recovery, a simultaneous decrease in ion leakage and disappearance of water soaking (symptoms of freeze-thaw injury) has been noted. Since reabsorption of ions during recovery must be an active process, recovery of plasma membrane ATPase (active transport system) functions has been implicated. In the present study, onion (Allium cepa L. cv Downing Yellow Globe) bulbs were subjected to a freeze-thaw stress which resulted in a reversible (recoverable) injury. Plasma membrane ATPase activity in the microsomes (isolated from the bulb scales) and ion leakage rate (efflux/hour) from the same scale tissue were measured immediately following thawing and after complete recovery. In injured tissue (30-40% water soaking), plasma membrane ATPase activity was reduced by about 30% and this was paralleled by about 25% higher ion leakage rate. As water soaking disappeared during recovery, the plasma membrane ATPase activity and the ion leakage rate returned to about the same level as the respective controls. Treatment of freeze-thaw injured tissue with vanadate, a specific inhibitor of plasma membrane ATPase, during postthaw prevented the recovery process. These results indicate that recovery of freeze-injured tissue depends on the functional activity of plasma membrane ATPase. PMID:16668063

  9. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

    OpenAIRE

    Laat, S.W. de; Tetteroo, P.A.T.; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van

    1984-01-01

    The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xaopus Levis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-(N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FP...

  10. The Role of the Plasma Membrane H+-ATPase in Plant Responses to Aluminum Toxicity

    Directory of Open Access Journals (Sweden)

    Jiarong Zhang

    2017-10-01

    Full Text Available Aluminum (Al toxicity is a key factor limiting plant growth and crop production on acid soils. Increasing the plant Al-detoxification capacity and/or breeding Al-resistant cultivars are a cost-effective strategy to support crop growth on acidic soils. The plasma membrane H+-ATPase plays a central role in all plant physiological processes. Changes in the activity of the plasma membrane H+-ATPase through regulating the expression and phosphorylation of this enzyme are also involved in many plant responses to Al toxicity. The plasma membrane H+-ATPase mediated H+ influx may be associated with the maintenance of cytosolic pH and the plasma membrane gradients as well as Al-induced citrate efflux mediated by a H+-ATPase-coupled MATE co-transport system. In particular, modulating the activity of plasma membrane H+-ATPase through application of its activators (e.g., magnesium or IAA or using transgenics has effectively enhanced plant resistance to Al stress in several species. In this review, we critically assess the available knowledge on the role of the plasma membrane H+-ATPase in plant responses to Al stress, incorporating physiological and molecular aspects.

  11. Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Liu, Zan; Xu, Bo; Nameta, Masaaki; Zhang, Ying; Magdeldin, Sameh; Yoshida, Yutaka; Yamamoto, Keiko; Fujinaka, Hidehiko; Yaoita, Eishin; Tasaki, Masayuki; Nakagawa, Yuki; Saito, Kazuhide; Takahashi, Kota; Yamamoto, Tadashi

    2013-06-01

    Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 μg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

  12. Simulations of simple linoleic acid-containing lipid membranes and models for the soybean plasma membranes.

    Science.gov (United States)

    Zhuang, Xiaohong; Ou, Anna; Klauda, Jeffery B

    2017-06-07

    The all-atom CHARMM36 lipid force field (C36FF) has been tested with saturated, monounsaturated, and polyunsaturated lipids; however, it has not been validated against the 18:2 linoleoyl lipids with an unsaturated sn-1 chain. The linoleoyl lipids are common in plants and the main component of the soybean membrane. The lipid composition of soybean plasma membranes has been thoroughly characterized with experimental studies. However, there is comparatively less work done with computational modeling. Our molecular dynamics (MD) simulation results show that the pure linoleoyl lipids, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0/18:2) and 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (di-18:2), agree very well with the experiments, which demonstrates the accuracy of the C36FF for the computational study of soybean membranes. Based on the experimental composition, the soybean hypocotyl and root plasma membrane models are developed with each containing seven or eight types of linoleoyl phospholipids and two types of sterols (sitosterol and stigmasterol). MD simulations are performed to characterize soybean membranes, and the hydrogen bonds and clustering results demonstrate that the lipids prefer to interact with the lipids of the same/similar tail unsaturation. All the results suggest that these two soybean membrane models can be used as a basis for further research in soybean and higher plant membranes involving membrane-associated proteins.

  13. Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

    Science.gov (United States)

    Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

    2015-10-20

    Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

  14. DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release.

    Directory of Open Access Journals (Sweden)

    Matthew E Pamenter

    Full Text Available During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra. Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs, which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed. DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA. Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS

  15. Interleaflet Coupling, Pinning, and Leaflet Asymmetry—Major Players in Plasma Membrane Nanodomain Formation

    Science.gov (United States)

    Fujimoto, Toyoshi; Parmryd, Ingela

    2017-01-01

    The plasma membrane has a highly asymmetric distribution of lipids and contains dynamic nanodomains many of which are liquid entities surrounded by a second, slightly different, liquid environment. Contributing to the dynamics is a continuous repartitioning of components between the two types of liquids and transient links between lipids and proteins, both to extracellular matrix and cytoplasmic components, that temporarily pin membrane constituents. This make plasma membrane nanodomains exceptionally challenging to study and much of what is known about membrane domains has been deduced from studies on model membranes at equilibrium. However, living cells are by definition not at equilibrium and lipids are distributed asymmetrically with inositol phospholipids, phosphatidylethanolamines and phosphatidylserines confined mostly to the inner leaflet and glyco- and sphingolipids to the outer leaflet. Moreover, each phospholipid group encompasses a wealth of species with different acyl chain combinations whose lateral distribution is heterogeneous. It is becoming increasingly clear that asymmetry and pinning play important roles in plasma membrane nanodomain formation and coupling between the two lipid monolayers. How asymmetry, pinning, and interdigitation contribute to the plasma membrane organization is only beginning to be unraveled and here we discuss their roles and interdependence. PMID:28119914

  16. Grafting of molecularly imprinted polymer to porous polyethylene filtration membranes by plasma polymerization.

    Science.gov (United States)

    Cowieson, D; Piletska, E; Moczko, E; Piletsky, S

    2013-08-01

    An application of plasma-induced grafting of polyethylene membranes with a thin layer of molecularly imprinted polymer (MIP) was presented. High-density polyethylene (HDPE) membranes, "Vyon," were used as a substrate for plasma grafting modification. The herbicide atrazine, one of the most popular targets of the molecular imprinting, was chosen as a template. The parameters of the plasma treatment were optimized in order to achieve a good balance between polymerization and ablation processes. Modified HDPE membranes were characterized, and the presence of the grafted polymeric layer was confirmed based on the observed weight gain, pore size measurements, and infrared spectrometry. Since there was no significant change in the porosity of the modified membranes, it was assumed that only a thin layer of the polymer was introduced on the surface. The experiments on the re-binding of the template atrazine to the membranes modified with MIP and blank polymers were performed. HDPE membranes which were grafted with polymer using continuous plasma polymerization demonstrated the best result which was expressed in an imprinted factor equal to 3, suggesting that molecular imprinting was successfully achieved.

  17. Interactions of Ras proteins with the plasma membrane and their roles in signaling.

    Science.gov (United States)

    Eisenberg, Sharon; Henis, Yoav I

    2008-01-01

    The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

  18. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    Science.gov (United States)

    Dormeyer, Wilma; van Hoof, Dennis; Mummery, Christine L; Krijgsveld, Jeroen; Heck, Albert J R

    2008-10-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological mechanisms that regulate proliferation and differentiation. The comparison of their membrane proteomes will help unravel the biological principles of pluripotency, and the identification of biomarker proteins in their plasma membranes is considered a crucial step to fully exploit pluripotent cells for therapeutic purposes. For these tasks, membrane proteomics is the method of choice, but as indicated by the scarce identification of membrane and plasma membrane proteins in global proteomic surveys it is not an easy task. In this minireview, we first describe the general challenges of membrane proteomics. We then review current sample preparation steps and discuss protocols that we found particularly beneficial for the identification of large numbers of (plasma) membrane proteins in human tumour- and embryo-derived stem cells. Our optimized assembled protocol led to the identification of a large number of membrane proteins. However, as the composition of cells and membranes is highly variable we still recommend adapting the sample preparation protocol for each individual system.

  19. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans.

    Science.gov (United States)

    Douglas, Lois M; Konopka, James B

    2016-03-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans.

  20. Plasma membrane organization promotes virulence of the human fungal pathogen Candida albicans

    Science.gov (United States)

    Douglas, Lois M.; Konopka, James. B.

    2017-01-01

    Candida albicans is a human fungal pathogen capable of causing lethal systemic infections. The plasma membrane plays key roles in virulence because it not only functions as a protective barrier, it also mediates dynamic functions including secretion of virulence factors, cell wall synthesis, invasive hyphal morphogenesis, endocytosis, and nutrient uptake. Consistent with this functional complexity, the plasma membrane is composed of a wide array of lipids and proteins. These components are organized into distinct domains that will be the topic of this review. Some of the plasma membrane domains that will be described are known to act as scaffolds or barriers to diffusion, such as MCC/eisosomes, septins, and sites of contact with the endoplasmic reticulum. Other zones mediate dynamic processes, including secretion, endocytosis, and a special region at hyphal tips that facilitates rapid growth. The highly organized architecture of the plasma membrane facilitates the coordination of diverse functions and promotes the pathogenesis of C. albicans. PMID:26920878

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

    International Nuclear Information System (INIS)

    Hayashi, Masami; Shimada, Yukiko; Inomata, Mitsushi; Ohno-Iwashita, Yoshiko

    2006-01-01

    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 β-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

  2. Plasma membrane lipids and their role in fungal virulence.

    Science.gov (United States)

    Rella, Antonella; Farnoud, Amir M; Del Poeta, Maurizio

    2016-01-01

    There has been considerable evidence in recent years suggesting that plasma membrane lipids are important regulators of fungal pathogenicity. Various glycolipids have been shown to impart virulent properties in several fungal species, while others have been shown to play a role in host defense. In addition to their role as virulence factors, lipids also contribute to other virulence mechanisms such as drug resistance, biofilm formation, and release of extracellular vesicles. In addition, lipids also affect the mechanical properties of the plasma membrane through the formation of packed microdomains composed mainly of sphingolipids and sterols. Changes in the composition of lipid microdomains have been shown to disrupt the localization of virulence factors and affect fungal pathogenicity. This review gathers evidence on the various roles of plasma membrane lipids in fungal virulence and how lipids might contribute to the different processes that occur during infection and treatment. Insight into the role of lipids in fungal virulence can lead to an improved understanding of the process of fungal pathogenesis and the development of new lipid-mediated therapeutic strategies. Published by Elsevier Ltd.

  3. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  4. Crystal structure of the plasma membrane proton pump

    DEFF Research Database (Denmark)

    Pedersen, Bjørn P.; Buch-Pedersen, Morten Jeppe; Morth, J. Preben

    2007-01-01

    A prerequisite for life is the ability to maintain electrochemical imbalances across biomembranes. In all eukaryotes the plasma membrane potential and secondary transport systems are energized by the activity of P-type ATPase membrane proteins: H1-ATPase (the proton pump) in plants and fungi1......-3, and Na1,K1-ATPase (the sodium-potassium pump) in animals4. The name P-type derives from the fact that these proteins exploit a phosphorylated reaction cycle intermediate of ATP hydrolysis5.The plasma membrane proton pumps belong to the type III P-type ATPase subfamily, whereas Na1,K1-ATPase and Ca21......- ATPase are type II6. Electron microscopy has revealed the overall shape of proton pumps7, however, an atomic structure has been lacking. Here we present the first structure of a P-type proton pump determined by X-ray crystallography. Ten transmembrane helices and three cytoplasmic domains define...

  5. Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

    International Nuclear Information System (INIS)

    Hasan, Nazarul; Hu, Chuan

    2010-01-01

    Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  6. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane

    Science.gov (United States)

    Saka, Sinem K.; Honigmann, Alf; Eggeling, Christian; Hell, Stefan W.; Lang, Thorsten; Rizzoli, Silvio O.

    2014-01-01

    Most proteins have uneven distributions in the plasma membrane. Broadly speaking, this may be caused by mechanisms specific to each protein, or may be a consequence of a general pattern that affects the distribution of all membrane proteins. The latter hypothesis has been difficult to test in the past. Here, we introduce several approaches based on click chemistry, through which we study the distribution of membrane proteins in living cells, as well as in membrane sheets. We found that the plasma membrane proteins form multi-protein assemblies that are long lived (minutes), and in which protein diffusion is restricted. The formation of the assemblies is dependent on cholesterol. They are separated and anchored by the actin cytoskeleton. Specific proteins are preferentially located in different regions of the assemblies, from their cores to their edges. We conclude that the assemblies constitute a basic mesoscale feature of the membrane, which affects the patterning of most membrane proteins, and possibly also their activity. PMID:25060237

  7. Apparatus for plasma surface treating and preparation of membrane layers

    NARCIS (Netherlands)

    1990-01-01

    An apparatus suitable for plasma surface treating (e.g., forming a membrane layer on a substrate surface) comprises a plasma generation section which is operable at least at substantially atmospheric pressure and is in communication via at least one plasma inlet (e.g., a nozzle) with an enclosed

  8. Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

    Science.gov (United States)

    Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

    2015-01-01

    Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

  9. Plasma-deposited hybrid silica membranes with a controlled retention of organic bridges

    Energy Technology Data Exchange (ETDEWEB)

    Ngamou, P.H.T.; Creatore, M. [Department of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven (Netherlands); Overbeek, J.P.; Kreiter, R.; Van Veen, H.M.; Vente, J.F. [ECN, Energy research Centre of the Netherlands, Petten (Netherlands); Wienk, I.M.; Cuperus, P.F. [SolSep BV, Apeldoorn (Netherlands)

    2013-03-05

    Hybrid organically bridged silica membranes are suitable for energy-efficient molecular separations under harsh industrial conditions. Such membranes can be useful in organic solvent nanofiltration if they can be deposited on flexible, porous and large area supports. Here, we report the proof of concept for applying an expanding thermal plasma to the synthesis of perm-selective hybrid silica films from an organically bridged monomer, 1,2-bis(triethoxysilyl)ethane. This membrane is the first in its class to be produced by plasma enhanced chemical vapor deposition. By tuning the plasma and process parameters, the organic bridging groups could be retained in the separating layer. This way, a defect free film could be made with pervaporation performances of an n-butanol-water mixture comparable with those of conventional ceramic supported membranes made by sol-gel technology (i.e. a water flux of [similar]1.8 kg m'-{sup 2} h{sup -1}, a water concentration in the permeate higher than 98% and a separation factor of >1100). The obtained results show the suitability of expanding thermal plasma as a technology for the deposition of hybrid silica membranes for molecular separations.

  10. 14C leucine chloromethylketone interaction with sarcoma 37 cell plasma membrane components

    International Nuclear Information System (INIS)

    Matthews, R.H.; Milo, G.E.; McMichael, T.L.; Lewis, N.J.

    1982-01-01

    Leucine chloromethylketone labelling of viable S37 cells was preferential for the plasma membrane fraction. The pattern of radiolabelling of the plasma membrane proteins was time-dependent. After 5 min the radiolabel was localized with glutamyl transpeptidase, and subsequently, with other physiologically active proteins as a function of time after incubation. Labelling of proteins was temperature-dependent and incubation of viable S37 cells with the radiolabelled substrate at 0 0 C yielded little or no radioactivity localized in the plasma membrane. The molecular weight of one radiolabelled substratemembrane protein complex was estimated on sodium dodecyl sulfate polyacrylamide gel electrophoresis to be between 100,000-200,000. (author)

  11. [Biocompatibility of poly-L-lactic acid/Bioglass-guided bone regeneration membranes processed with oxygen plasma].

    Science.gov (United States)

    Fang, Wei; Zeng, Shu-Guang; Gao, Wen-Feng

    2015-04-01

    To prepare and characterize a nano-scale fibrous hydrophilic poly-L-lactic acid/ Bioglass (PLLA/BG) composite membrane and evaluate its biocompatibility as a composite membrane for guiding bone regeneration (GBR). PLLA/BG-guided bone regeneration membrane was treated by oxygen plasma to improved its hydrophilicity. The growth of MG-63 osteoblasts on the membrane was observed using Hoechst fluorescence staining, and the biocompatibility of the membrane was evaluated by calculating the cells adhesion rate and proliferation rate. Osteogenesis of MG-63 cells was assessed by detecting alkaline phosphatase (ALP), and the formation of calcified nodules and cell morphology changes were observed using scanning electron microscope (SEM). The cell adhesion rates of PLLA/BG-guided bone regeneration membrane treated with oxygen plasma were (30.570±0.96)%, (47.27±0.78)%, and (66.78±0.69)% at 1, 3, and 6 h, respectively, significantly higher than those on PLLA membrane and untreated PLLA/BG membrane (Pmembranes increased with time, but highest on oxygen plasma-treated PLLA/BG membrane (Pplasma treatment of the PLLA/BG membrane promoted cell adhesion. The membranes with Bioglass promoted the matrix secretion of the osteoblasts. Under SEM, the formation of calcified nodules and spindle-shaped cell morphology were observed on oxygen plasma-treated PLLA/BG membrane. Oxygen plasma-treated PLLA/BG composite membrane has good biocompatibility and can promote adhesion, proliferation and osteogenesis of the osteoblasts.

  12. Surface characterization of the chitosan membrane after oxygen plasma treatment and its aging effect

    International Nuclear Information System (INIS)

    Wang Yingjun; Yin Shiheng; Ren Li; Zhao Lianna

    2009-01-01

    Chitosan has received considerable attention for biomedical applications in recent years because of its biocompatibility and biodegradability. In this paper, angle-resolved x-ray photoelectron spectroscopy (ARXPS) was carried out to investigate the chemical groups' spatial orientation on the chitosan membrane surface. Oxygen plasma treatment was also employed to improve the surface hydrophilicity of the chitosan membrane. The results of ARXPS revealed the distribution of surface polar groups, such as-OH and O=CNH 2 toward the membrane bulk, which was the origin of the chitosan membrane surface hydrophobicity. The contact angle measurements and XPS results indicated that oxygen plasma treatment can markedly improve the surface hydrophilicity and surface energy of the chitosan membrane by incorporating oxygen-containing polar groups. With the existence of the aging process, the influence of plasma treatment was not permanent, it faded with storage time. The ARXPS result discovered that the reorientation of polar functional groups generated by plasma treatment toward the membrane bulk was primarily responsible for the aging effect.

  13. Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

    Science.gov (United States)

    Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Imaoka, Yuka; Itagaki, Yoshitsuna; Suzuki, Yuki

    2018-01-01

    Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. PMID:29723197

  14. Distribution of IGF receptors in the plasma membrane of proximal tubular cells

    International Nuclear Information System (INIS)

    Hammerman, M.R.; Rogers, S.

    1987-01-01

    To characterize the distribution of receptors for insulin-like growth factors I and II (IGF I and II) in the plasma membrane of the renal proximal tubular cell, the authors measured binding of 125 I-labeled IGF I and 125 I-labeled IGF II to proximal tubular basolateral and brush-border membranes and characterized IGF I-stimulated phosphorylation of detergent-solubilized membranes. 125 I-IGF bound primarily to a 135,000 relative molecular weight (M r ) protein and IGF II to a 260,000 M r protein in isolated membranes. Binding of 125 I-IGF I was severalfold greater in basolateral than in brush-border membranes. IGF I-stimulated phosphorylation of the 92,000 M r β-subunit of its receptors could be demonstrated only in basolateral membranes. These findings are consistent with an asymmetrical distribution of receptors for IGF I in the plasma membrane of the renal proximal tubular cell, localization being primary on the basolateral side. In contrast, binding of 125 I-IGF II to isolated basolateral and brush-border membranes was equivalent, suggesting that receptors for this peptide are distributed more symmetrically in the plasma membrane. The findings suggest that the action of IGF I in proximal tubule are mediated via interaction of circulating peptide with specific receptors in the basolateral membrane. However, the findings established the potential for actions of IGF II to be exerted in proximal tubule via interaction with both basolateral and/or brush-border membrane receptors

  15. (poly)Phosphoinositide phosphorylation is a marker for plasma membrane in Friend erythroleukaemic cells

    NARCIS (Netherlands)

    Rawyler, A.J.; Roelofsen, B.; Wirtz, K.W.A.; Kamp, J.A.F. op den

    1982-01-01

    Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the

  16. Detecting subtle plasma membrane perturbation in living cells using second harmonic generation imaging.

    Science.gov (United States)

    Moen, Erick K; Ibey, Bennett L; Beier, Hope T

    2014-05-20

    The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ~50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. There Is No Simple Model of the Plasma Membrane Organization

    Czech Academy of Sciences Publication Activity Database

    de la serna, J. B.; Schütz, G.; Eggeling, Ch.; Cebecauer, Marek

    2016-01-01

    Roč. 4, SEP 2016 (2016), 106 ISSN 2296-634X R&D Projects: GA ČR GA15-06989S Institutional support: RVO:61388955 Keywords : plasma membrane * membrane organization models * heterogeneous distribution Subject RIV: CF - Physical ; Theoretical Chemistry

  18. The C-terminal domain of TRPV4 is essential for plasma membrane localization.

    Science.gov (United States)

    Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

    2008-02-01

    Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

  19. Endoplasmic Reticulum-Plasma Membrane Contact Sites.

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2017-06-20

    The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

  20. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    Science.gov (United States)

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  1. The cell-based L-glutathione protection assays to study endocytosis and recycling of plasma membrane proteins.

    Science.gov (United States)

    Cihil, Kristine M; Swiatecka-Urban, Agnieszka

    2013-12-13

    Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

  2. Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

    Science.gov (United States)

    Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

    2013-08-06

    Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

  3. Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

    Science.gov (United States)

    Correani, Virginia; Di Francesco, Laura; Mignogna, Giuseppina; Fabrizi, Cinzia; Leone, Stefano; Giorgi, Alessandra; Passeri, Alessia; Casata, Roberto; Fumagalli, Lorenzo; Maras, Bruno; Schininà, M Eugenia

    2017-09-01

    In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

    Science.gov (United States)

    Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

    2014-03-21

    A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

  5. Surface-enhanced Raman scattering reveals adsorption of mitoxantrone on plasma membrane of living cells

    International Nuclear Information System (INIS)

    Breuzard, G.; Angiboust, J.-F.; Jeannesson, P.; Manfait, M.; Millot, J.-M.

    2004-01-01

    Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 deg. C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells

  6. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast

    International Nuclear Information System (INIS)

    Fisher, G.J.; Kelley, L.K.; Smith, C.H.

    1987-01-01

    As a first step in understanding the cellular basis of maternal-fetal calcium transfer, the authors examined the characteristics of calcium uptake by a highly purified preparation of the syncytiotrophoblast basal (fetal facing) plasma membrane. In the presence of nanomolar concentrations of free calcium, basal membranes demonstrated substantial ATP-dependent calcium uptake. This uptake required magnesium, was not significantly affected by Na + or K + (50 mM), or sodium azide (10 mM). Intravesicular calcium was rapidly and completely released by the calcium ionophore rapidly and completely released by the calcium ionophore A23187. Calcium transport was significantly stimulated by the calcium-dependent regulatory protein calmodulin. Placental membrane fractions enriched in endoplasmic reticulum (ER) and mitochondria also demonstrated ATP-dependent calcium uptake. In contrast to basal membrane, mitochondrial calcium uptake was completely inhibited by azide. The rate of calcium uptake was completely inhibited by azide. The rate of calcium uptake by the ER was only 20% of that of basal membranes. They conclude that the placental basal plasma membrane possesses a high-affinity calcium transport system similar to that found in plasma membranes of a variety of cell types. This transporter is situated to permit it to function in vivo in maternal-fetal calcium transfer

  7. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    International Nuclear Information System (INIS)

    Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7- 3 H]IAA([ 3 H]N 3 IAA), in a manner similar to the accumulation of [ 3 H]IAA. The association of the [ 3 H]N 3 IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [ 3 H]N 3 IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO 4 /PAGE and fluorography. When the reaction temperature was lowered to -196 degree C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors

  8. Molecular cloning and characterization of the plasma membrane ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... Southern blot analysis indicated that OvPIP gene was present in O. ... Key words: Orychophragmus violaceus, plasma membrane, tonoplast aquaporins .... fractionated by 0.8% agarose gel electrophoresis and transferred to.

  9. Pretreatment and Membrane Hydrophilic Modification to Reduce Membrane Fouling

    Directory of Open Access Journals (Sweden)

    Huaqiang Chu

    2013-09-01

    Full Text Available The application of low pressure membranes (microfiltration/ultrafiltration has undergone accelerated development for drinking water production. However, the major obstacle encountered in its popularization is membrane fouling caused by natural organic matter (NOM. This paper firstly summarizes the two factors causing the organic membrane fouling, including molecular weight (MW and hydrophilicity/hydrophobicity of NOM, and then presents a brief introduction of the methods which can prevent membrane fouling such as pretreatment of the feed water (e.g., coagulation, adsorption, and pre-oxidation and membrane hydrophilic modification (e.g., plasma modification, irradiation grafting modification, surface coating modification, blend modification, etc.. Perspectives of further research are also discussed.

  10. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

    Science.gov (United States)

    Leser, George P; Lamb, Robert A

    2017-05-01

    Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

  11. [Does a lateral gradient of membrane potential on the plasma membrane of growing pollen tube of germinating pollen grain exist?].

    Science.gov (United States)

    Andreev, I M

    2011-01-01

    The data presented in the article by Breigina et al. (2009) "Changes in the membrane potential during pollen grain germination and pollen tube growth" (Tsitologiya. 51 (10): 815-823) and concerning the measurement of electric membrane potential (Delta Psi) on the plasma membrane of growing pollen tube of germinating pollen grain with the use of fluorescent potential-sensitive dye, di-4-ANEPPS, were critically analyzed in order to clarify whether a lateral gradient of Delta Psi on this membrane indeed exists. This analysis showed that the main conclusion of the authors of the above article on the existence of polar distribution of Delta Psi along the pollen tube plasma membrane is not in accordance with a number of known peculiarities of di-4-ANEPPS behavior in biological membranes and requires a significant revision. The findings in question reported by the authors, in my opinion, might be interpreted as evidence for the presence on the plasma membrane of growing pollen tube not only the membrane potential Delta Psi but also lateral gradient of so called intra-membrane dipole potential. Based on the comments made, another interpretation of the experimental results described by Breigina et al. has been offered. In addition, some drawbacks in the methodology used by the authors for measurement of Delta Psi with other fluorescent potential-sensitive dye, DiBAC3(3), are also shortly considered.

  12. Possible evidence that dehydroepiandrosterone sulfate (DHA-S) stimulates cervical ripening by a membrane-mediated process: Specific binding-sites in plasma membrane from human uterine cervix

    International Nuclear Information System (INIS)

    Ohno, T.; Imai, A.; Tamaya, T.

    1991-01-01

    Fetal adrenal steroid, dehydroepiandrosterone sulfate (DHA-S) is well known to promote cervical ripening in late pregnancy. The presence of sites specifically binding the DHA-S in plasma membrane was studied in human cervical fibroblasts prepared from pregnant uterus. The fibroblasts were incubated with 3 H DHA-S and then fractionated into plasma membranes, cytosol, nuclei, and other organella debris. The specific activity of 3H-count in the plasma membrane fraction was enriched ∼ 7-fold compared with the whole homogenate. When the isolated plasma membrane preparations from the fibroblasts were exposed to 3 H DHA-S, the binding showed saturation kinetics; an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) of 1.25 pmol/mg protein. The present results suggest that DHA is bound to and recognized by components in plasma membrane, and may exert its action on cervical ripening through the membrane-mediated processes

  13. Receptor kinase-mediated control of primary active proton pumping at the plasma membrane

    DEFF Research Database (Denmark)

    Fuglsang, Anja Thoe; Kristensen, Astrid; Cuin, Tracey A.

    2014-01-01

    Acidification of the cell wall space outside the plasma membrane is required for plant growth and is the result of proton extrusion by the plasma membrane-localized H+-ATPases. Here we show that the major plasma membrane proton pumps in Arabidopsis, AHA1 and AHA2, interact directly in vitro...... and in planta with PSY1R, a receptor kinase of the plasma membrane that serves as a receptor for the peptide growth hormone PSY1. The intracellular protein kinase domain of PSY1R phosphorylates AHA2/AHA1 at Thr-881, situated in the autoinhibitory region I of the C-terminal domain. When expressed in a yeast...... heterologous expression system, the introduction of a negative charge at this position caused pump activation. Application of PSY1 to plant seedlings induced rapid in planta phosphorylation at Thr-881, concomitant with an instantaneous increase in proton efflux from roots. The direct interaction between AHA2...

  14. Rapid, directed transport of DC-SIGN clusters in the plasma membrane.

    Science.gov (United States)

    Liu, Ping; Weinreb, Violetta; Ridilla, Marc; Betts, Laurie; Patel, Pratik; de Silva, Aravinda M; Thompson, Nancy L; Jacobson, Ken

    2017-11-01

    C-type lectins, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 μm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor-driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions.

  15. Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy.

    Science.gov (United States)

    Trendowski, Matthew; Fondy, Thomas P

    2015-08-01

    Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

  16. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

    Science.gov (United States)

    Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

    2016-12-06

    Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

  17. Purification of a large molecular weight transglutaminase substrate from liver plasma membranes

    International Nuclear Information System (INIS)

    Slife, C.W.; Morris, G.S.; Tyrrell, D.J.

    1986-01-01

    Transglutaminases are enzymes which catalyze the covalent crosslinking of proteins by forming epsilon(γ-glutamyl)lysine isopeptide linkages. In earlier studies, the authors reported that a large molecular weight protein aggregate in rat liver plasma membranes served as a substrate for a plasma membrane-associated transglutaminase. The enzyme specifically incorporated a lysine analog, [ 3 H]putrescine, into a protein complex which remained at the top of an acrylamide gel upon electrophoresis in SDS and reducing agents. The complex has now been isolated by extracting the plasma membranes with detergent (octylglucoside) resuspending the detergent insoluble residues in 6 M guanidine HCl and chromatographing the residue on a 4% agarose column in 6 M guanidine HCl. Most of the radioactivity is found in the void volume fractions from the column. SDS polyacrylamide gel electrophoresis shows that these fractions contain mostly proteins that do not enter the acrylamide gel. Since this purification procedure is essentially the same as that used to isolate a rat hepatocyte adhesion factor from rat liver plasma membranes it is possible that the large molecular weight transglutaminase substrate and the adhesion factor are contained in the same protein aggregate

  18. Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

    Science.gov (United States)

    Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

    2014-01-01

    The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

  19. Plasma-chemical modification of the structure and properties of poly(ethylene terephthalate) track membranes

    International Nuclear Information System (INIS)

    Kravets, L I; Dmitriev, S N; Dinescu, G; Lazea, A; Sleptsov, V V; Elinson, V M

    2007-01-01

    A process of extraction of the low-molecular products of the synthesis from the poly(ethylene terephthalate) track membranes modified by plasma has been investigated. It is shown that the deposition of a thin polymeric hydrocarbon film by cyclohexane plasma on the membrane surface with preliminary treatment in a plasma of non-polymerizing gases, for example oxygen, allows one to produce membranes possessing a high productivity. Their advantages are much better hydrodynamic properties and a small amount of the low-molecular products of the synthesis extracted by organic solvents

  20. Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

    DEFF Research Database (Denmark)

    Zech, Tobias; Ejsing, Christer S.; Gaus, Katharina

    2009-01-01

    Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin...... and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation...... domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate...

  1. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    Science.gov (United States)

    Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

    2014-11-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

    2013-01-01

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  3. Assembly of fission yeast eisosomes in the plasma membrane of budding yeast: Import of foreign membrane microdomains

    Czech Academy of Sciences Publication Activity Database

    Vaškovičová, Katarína; Strádalová, Vendula; Efenberk, Aleš; Opekarová, Miroslava; Malínský, Jan

    2015-01-01

    Roč. 94, č. 1 (2015), s. 1-11 ISSN 0171-9335 R&D Projects: GA ČR(CZ) GAP302/11/0146 Institutional support: RVO:68378041 Keywords : plasma membrane * membrane microdomain * MCC Subject RIV: EA - Cell Biology Impact factor: 4.011, year: 2015

  4. Radio-iodination of plasma membranes of toad bladder epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, H J; Edelman, I S [California Univ., San Francisco (USA). Cardiovascular Research Inst.; California Univ., San Francisco (USA). Dept. of Medicine; California Univ., San Francisco (USA). Dept. of Biochemistry and Biophysics)

    1979-01-01

    The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium with /sup 125/I-Na, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labelling, was attained after about 30 min of exposure of the intact bladder to the labelling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT). (orig./AJ).

  5. Remodeling of the postsynaptic plasma membrane during neural development.

    Science.gov (United States)

    Tulodziecka, Karolina; Diaz-Rohrer, Barbara B; Farley, Madeline M; Chan, Robin B; Di Paolo, Gilbert; Levental, Kandice R; Waxham, M Neal; Levental, Ilya

    2016-11-07

    Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. © 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  6. Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment.

    Directory of Open Access Journals (Sweden)

    Yvonne S Ziegler

    Full Text Available The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

  7. Interactions of sugar-based bolaamphiphiles with biomimetic systems of plasma membranes.

    Science.gov (United States)

    Nasir, Mehmet Nail; Crowet, Jean-Marc; Lins, Laurence; Obounou Akong, Firmin; Haudrechy, Arnaud; Bouquillon, Sandrine; Deleu, Magali

    2016-11-01

    Glycolipids constitute a class of molecules with various biological activities. Among them, sugar-based bolaamphiphiles characterized by their biocompatibility, biodegradability and lower toxicity, became interesting for the development of efficient and low cost lipid-based drug delivery systems. Their activity seems to be closely related to their interactions with the lipid components of the plasma membrane of target cells. Despite many works devoted to the chemical synthesis and characterization of sugar-based bolaamphiphiles, their interactions with plasma membrane have not been completely elucidated. In this work, two sugar-based bolaamphiphiles differing only at the level of their sugar residues were chemically synthetized. Their interactions with membranes have been investigated using model membranes containing or not sterol and with in silico approaches. Our findings indicate that the nature of sugar residues has no significant influence for their membrane interacting properties, while the presence of sterol attenuates the interactions of both bolaamphiphiles with the membrane systems. The understanding of this distinct behavior of bolaamphiphiles towards sterol-containing membrane systems could be useful for their applications as drug delivery systems. Copyright © 2016. Published by Elsevier B.V.

  8. Parallel artificial liquid membrane extraction of acidic drugs from human plasma

    DEFF Research Database (Denmark)

    Roldan-Pijuan, Mercedes; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2015-01-01

    The new sample preparation concept “Parallel artificial liquid membrane extraction (PALME)” was evaluated for extraction of the acidic drugs ketoprofen, fenoprofen, diclofenac, flurbiprofen, ibuprofen, and gemfibrozil from human plasma samples. Plasma samples (250 μL) were loaded into individual......-performance liquid chromatography-ultraviolet detection of the individual acceptor solutions. Important PALME parameters including the chemical composition of the liquid membrane, extraction time, and sample pH were optimized, and the extraction performance was evaluated. Except for flurbiprofen, exhaustive...

  9. Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

    Science.gov (United States)

    Löw, Hans; Crane, Frederick L; Morré, D James

    2012-11-01

    The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Production of selective membranes using plasma deposited nanochanneled thin films

    Directory of Open Access Journals (Sweden)

    Rodrigo Amorim Motta Carvalho

    2006-12-01

    Full Text Available The hydrolization of thin films obtained by tetraethoxysilane plasma polymerization results in the formation of a nanochanneled silicone like structure that could be useful for the production of selective membranes. Therefore, the aim of this work is to test the permeation properties of hydrolyzed thin films. The films were tested for: 1 permeation of polar organic compounds and/or water in gaseous phase and 2 permeation of salt in liquid phase. The efficiency of permeation was tested using a quartz crystal microbalance (QCM technique in gas phase and conductimetric analysis (CA in liquid phase. The substrates used were: silicon for characterization of the deposited films, piezoelectric quartz crystals for tests of selective membranes and cellophane paper for tests of permeation. QCM analysis showed that the nanochannels allow the adsorption and/or permeation of polar organic compounds, such as acetone and 2-propanol, and water. CA showed that the films allow salt permeation after an inhibition time needed for hydrolysis of the organic radicals within the film. Due to their characteristics, the films can be used for grains protection against microorganism proliferation during storage without preventing germination.

  11. Magnetic apatite for structural insights on the plasma membrane

    International Nuclear Information System (INIS)

    Stanca, Sarmiza E; Müller, Robert; Dellith, Jan; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph

    2015-01-01

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications. (paper)

  12. Magnetic apatite for structural insights on the plasma membrane

    Science.gov (United States)

    Stanca, Sarmiza E.; Müller, Robert; Dellith, Jan; Nietzsche, Sandor; Stöckel, Stephan; Biskup, Christoph; Deckert, Volker; Krafft, Christoph; Popp, Jürgen; Fritzsche, Wolfgang

    2015-01-01

    The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

  13. The molecular mechanisms of plant plasma membrane intrinsic proteins trafficking and stress response.

    Science.gov (United States)

    Wang, Xing; Zhang, Ji-long; Feng, Xiu-xiu; Li, Hong-jie; Zhang, Gen-fa

    2017-04-20

    Plasma membrane intrinsic proteins (PIPs) are plant channel proteins located on the plasma membrane. PIPs transfer water, CO 2 and small uncharged solutes through the plasma membrane. PIPs have high selectivity to substrates, suggestive of a central role in maintaining cellular water balance. The expression, activity and localization of PIPs are regulated at the transcriptional and post-translational levels, and also affected by environmental factors. Numerous studies indicate that the expression patterns and localizations of PIPs can change in response to abiotic stresses. In this review, we summarize the mechanisms of PIP trafficking, transcriptional and post-translational regulations, and abiotic stress responses. Moreover, we also discuss the current research trends and future directions on PIPs.

  14. The Enzymology of Protein Translocation across the Escherichia coli Plasma Membrane

    NARCIS (Netherlands)

    Wickner, William; Driessen, Arnold J.M.; Hartl, Franz-Ulrich

    1991-01-01

    Converging physiological, genetic, and biochemical studies have established the salient features of preprotein translocation across the plasma membrane of Escherichia coli. Translocation is catalyzed by two proteins, a soluble chaperone and a membrane-bound translocase. SecB, the major chaperone for

  15. Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

    2016-05-01

    Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

  16. Apparatus suitable for plasma surface treating and process for preparing membrane layers

    NARCIS (Netherlands)

    1988-01-01

    The invention relates to an apparatus suitable for plasma surface treating (e.g. forming a membrane layer on a substrate) which comprises a plasma generation section (2) which is in communication via at least one plasma inlet means (4) (e.g. a nozzle) with an enclosed plasma treating section (3)

  17. GLUT-4 content in plasma membrane of muscle from patients with non-insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Lund, S; Vestergaard, H; Andersen, P H

    1993-01-01

    The abundance of GLUT-4 protein in both total crude membrane and plasma membrane fractions of vastus lateralis muscle from 13 obese non-insulin-dependent diabetes mellitus (NIDDM) patients and 14 healthy subjects were examined in the fasting state and after supraphysiological hyperinsulinemia....... In the basal state the immunoreactive mass of GLUT-4 protein both in the crude membrane preparation and in the plasma membrane fraction was similar in NIDDM patients and control subjects. Moreover, in vivo insulin exposure neither for 30 min nor for 4 h had any impact on the content of GLUT-4 protein in plasma...... membranes. With the use of the same methodology, antibody, and achieving the same degree of plasma membrane purification and recovery, we found, however, that intraperitoneal administration of insulin to 7-wk-old rats within 30 min increased the content of GLUT-4 protein more than twofold (P

  18. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    Science.gov (United States)

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  19. The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane.

    Science.gov (United States)

    Spang, Anne

    2015-03-10

    The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed.

  20. In vitro sealing of iatrogenic fetal membrane defects by a collagen plug imbued with fibrinogen and plasma.

    Science.gov (United States)

    Engels, A C; Hoylaerts, M F; Endo, M; Loyen, S; Verbist, G; Manodoro, S; DeKoninck, P; Richter, J; Deprest, J A

    2013-02-01

    We aimed to demonstrate local thrombin generation by fetal membranes, as well as its ability to generate fibrin from fibrinogen concentrate. Furthermore, we aimed to investigate the efficacy of collagen plugs, soaked with plasma and fibrinogen, to seal iatrogenic fetal membrane defects. Thrombin generation by homogenized fetal membranes was measured by calibrated automated thrombography. To identify the coagulation caused by an iatrogenic membrane defect, we analyzed fibrin formation by optical densitometry, upon various concentrations of fibrinogen. The ability of a collagen plug soaked with fibrinogen and plasma was tested in an ex vivo model for its ability to seal an iatrogenic fetal membrane defect. Fetal membrane homogenates potently induced thrombin generation in amniotic fluid and diluted plasma. Upon the addition of fibrinogen concentrate, potent fibrin formation was triggered. Measured by densiometry, fibrin formation was optimal at 1250 µg/mL fibrinogen in combination with 4% plasma. A collagen plug soaked with fibrinogen and plasma sealed an iatrogenic membrane defect about 35% better than collagen plugs without these additives (P = 0.037). These in vitro experiments suggest that the addition of fibrinogen and plasma may enhance the sealing efficacy of collagen plugs in closing iatrogenic fetal membrane defects. © 2013 John Wiley & Sons, Ltd.

  1. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.

    1986-01-01

    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  2. Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

    Science.gov (United States)

    Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

    2018-05-01

    In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

  3. Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

    Science.gov (United States)

    Gerstle, Zoe; Desai, Rohan; Veatch, Sarah L

    2018-01-01

    Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy. © 2018 Elsevier Inc. All rights reserved.

  4. LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

    Science.gov (United States)

    Clayton, R N; Shakespear, R A; Marshall, J C

    1978-06-01

    Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

  5. Dynamic complexity: plant receptor complexes at the plasma membrane.

    Science.gov (United States)

    Burkart, Rebecca C; Stahl, Yvonne

    2017-12-01

    Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Derangement of cellular plasma membranes due to non-lethal radiation doses

    International Nuclear Information System (INIS)

    Koeteles, G.J.; Kubasova, T.; Somosy, Z.; Horvath, L.

    1983-01-01

    Earlier observations in the laboratory on fibroblasts and various blood cells of animal and human origins pointed to alteration of concanavalin A binding sites of plasma membranes as well as to concomitant morphological changes and scanning electron microscopic appearance of cell surfaces following sub-lethal doses of X-, fission neutron and beta irradiations. The effects appeared early and existed temporarily; their intensities and the restitution of membrane function depended on radiation doses, types and conditions of cells. In the present paper further aspects of structural and functional derangements of plasma membranes are introduced which were provoked by X- and tritium beta irradiation in the dose range up to 2.5 Gy and in the concentration range from 3.7 kBq/mL, respectively. The state of membrane structure was followed by bindings of various ligands of different receptor requirements, concanavalin A, cationized ferritin and polio virus. In the case of X-irradiation the binding conditions suggest the shift of overall negative surface charges to less negative ones. It was also found that radiation-induced phenomena appear on the cell surface unevenly. Long- and short-term treatments of cells with 3 H-thymidine and 3 H-water also perturb the plasma membrane; beta irradiation affects it directly. Membrane structure and function are suggested to offer good biological models to study correlation of energy deposition and biological effects, both restricted to domains of nanometre range. The data give evidence for radiation-induced membrane alterations in the sub-lethal or non-lethal ranges which might have consequences in the development of stochastic and non-stochastic effects. (author)

  7. Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution

    International Nuclear Information System (INIS)

    Sasaki, Shota; Honda, Ryosuke; Hokari, Yutaro; Takashima, Keisuke; Kaneko, Toshiro; Kanzaki, Makoto

    2016-01-01

    Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor( s ), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OH aq ), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (<1 mm), and the plasma-produced OH aq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OH aq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OH aq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool. (paper)

  8. Organization and Dynamics of Receptor Proteins in a Plasma Membrane.

    Science.gov (United States)

    Koldsø, Heidi; Sansom, Mark S P

    2015-11-25

    The interactions of membrane proteins are influenced by their lipid environment, with key lipid species able to regulate membrane protein function. Advances in high-resolution microscopy can reveal the organization and dynamics of proteins and lipids within living cells at resolutions membranes of in vivo-like complexity. We explore the dynamics of proteins and lipids in crowded and complex plasma membrane models, thereby closing the gap in length and complexity between computations and experiments. Our simulations provide insights into the mutual interplay between lipids and proteins in determining mesoscale (20-100 nm) fluctuations of the bilayer, and in enabling oligomerization and clustering of membrane proteins.

  9. Performance enhancement of membrane electrode assemblies with plasma etched polymer electrolyte membrane in PEM fuel cell

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Yong-Hun; Yoon, Won-Sub [School of Advanced Materials Engineering, Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul 136-702 (Korea); Bae, Jin Woo; Cho, Yoon-Hwan; Lim, Ju Wan; Ahn, Minjeh; Jho, Jae Young; Sung, Yung-Eun [World Class University (WCU) program of Chemical Convergence for Energy and Environment (C2E2), School of Chemical and Biological Engineering, College of Engineering, Seoul National University (SNU), 599 Gwanak-Ro, Gwanak-gu, Seoul 151-744 (Korea); Kwon, Nak-Hyun [Fuel Cell Vehicle Team 3, Advanced Technology Center, Corporate Research and Development Division, Hyundai-Kia Motors, 104 Mabuk-dong, Giheung-gu, Yongin-si, Gyeonggi-do 446-912 (Korea)

    2010-10-15

    In this work, a surface modified Nafion 212 membrane was fabricated by plasma etching in order to enhance the performance of a membrane electrode assembly (MEA) in a polymer electrolyte membrane fuel cell. Single-cell performance of MEA at 0.7 V was increased by about 19% with membrane that was etched for 10 min compared to that with untreated Nafion 212 membrane. The MEA with membrane etched for 20 min exhibited a current density of 1700 mA cm{sup -2} at 0.35 V, which was 8% higher than that of MEA with untreated membrane (1580 mA cm{sup -2}). The performances of MEAs containing etched membranes were affected by complex factors such as the thickness and surface morphology of the membrane related to etching time. The structural changes and electrochemical properties of the MEAs with etched membranes were characterized by field emission scanning electron microscopy, Fourier transform-infrared spectrometry, electrochemical impedance spectroscopy, and cyclic voltammetry. (author)

  10. Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes.

    Science.gov (United States)

    Bacakova, Marketa; Lopot, Frantisek; Hadraba, Daniel; Varga, Marian; Zaloudkova, Margit; Stranska, Denisa; Suchy, Tomas; Bacakova, Lucie

    2015-01-01

    It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  11. Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

    Science.gov (United States)

    Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

    2014-03-01

    Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Further characterization of the red beet plasma membrane Ca2+-ATPase using GTP as an alternative substrate

    International Nuclear Information System (INIS)

    Williams, L.E.; Schueler, S.B.; Briskin, D.P.

    1990-01-01

    The GTP-driven component of Ca 2+ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca 2+ -translocating ATPase and assess its utility as a probe for this transport system. Uptake of 45 Ca 2+ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca 2+ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca 2+ -ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of 45 Ca 2+ -uptake by exogeneous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven 45 Ca 2+ uptake represents the capacity of the plasma membrane Ca 2+ -translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ- 32 P]GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca 2+ -ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca 2+ -ATPases present in animal cells

  13. Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

    International Nuclear Information System (INIS)

    Shibata, Y.; Abiko, Y.; Ohno, H.; Araki, T.; Takiguchi, H.

    1988-01-01

    Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A 2 had an optimum pH at 4.5, and enzyme activation was observed in Ca ++ -free medium; but the maximum activity was found at 0.5 mM Ca ++ concentration. The Km value for PC of acidic PLase A 2 was 4.2 μM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A 2 in light of the uncompetitive manner of Ca ++ action. Furthermore, the release of [ 3 H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca ++ at pH 4.5. These data suggest that the acid PLase A 2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A 2 properties are opposite to those found for lysosomal PLase A 2

  14. Epithelial cell-cell junctions and plasma membrane domains

    NARCIS (Netherlands)

    Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

    Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

  15. Glycine transporter dimers: evidence for occurrence in the plasma membrane

    DEFF Research Database (Denmark)

    Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette

    2008-01-01

    membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2......Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma...

  16. Influence of plasma modification on hygienic properties of textile fabrics with nonporous membrane coating

    Science.gov (United States)

    Voznesensky, E. F.; Ibragimov, R. G.; Vishnevskaya, O. V.; Sisoev, V. A.; Lutfullina, G. G.; Tihonova, N. V.

    2017-11-01

    The work investigated the possibility of using plasma modification to improve the hygienic properties of textile materials with nonporous membrane coating to improve vapor-, air-permeability and water-resistant. Determined that, after plasma modification changes degree of supramolecular orderliness of the polymers nonporous membrane coating and the base fabric.

  17. Development of Polysulfone Hollow Fiber Porous Supports for High Flux Composite Membranes: Air Plasma and Piranha Etching

    Directory of Open Access Journals (Sweden)

    Ilya Borisov

    2017-02-01

    Full Text Available For the development of high efficiency porous supports for composite membrane preparation, polysulfone (PSf hollow fiber membranes (outer diameter 1.57 mm, inner diameter 1.12 mm were modified by air plasma using the low temperature plasma treatment pilot plant which is easily scalable to industrial level and the Piranha etch (H2O2 + H2SO4. Chemical and plasma modification affected only surface layers and did not cause PSf chemical structure change. The modifications led to surface roughness decrease, which is of great importance for further thin film composite (TFC membranes fabrication by dense selective layer coating, and also reduced water and ethylene glycol contact angle values for modified hollow fibers surface. Furthermore, the membranes surface energy increased two-fold. The Piranha mixture chemical modification did not change the membranes average pore size and gas permeance values, while air plasma treatment increased pore size 1.5-fold and also 2 order enhanced membranes surface porosity. Since membranes surface porosity increased due to air plasma treatment the modified membranes were used as efficient supports for preparation of high permeance TFC membranes by using poly[1-(trimethylsilyl-1-propyne] as an example for selective layer fabrication.

  18. Preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient.

    Science.gov (United States)

    Bermejo, Marie Kristel; Milenkovic, Marija; Salahpour, Ali; Ramsey, Amy J

    2014-09-03

    Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960's, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

  19. Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics.

    Directory of Open Access Journals (Sweden)

    Hadil F Al-Jallad

    2011-01-01

    Full Text Available Transglutaminase activity, arising potentially from transglutaminase 2 (TG2 and Factor XIIIA (FXIIIA, has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.

  20. Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

    Science.gov (United States)

    Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

    2017-01-01

    Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

  1. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

    International Nuclear Information System (INIS)

    Funakoshi, Takeshi; Yanai, Akie; Shinoda, Koh; Kawano, Michio M.; Mizukami, Yoichi

    2006-01-01

    Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain

  2. A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

    Science.gov (United States)

    Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

    1997-04-29

    Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

  3. Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

    Science.gov (United States)

    Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

    2017-02-01

    palmitoylation of TF regulates its localization to the plasma membrane, which is the site of alphavirus budding. Mutants in which TF is not palmitoylated display drastically reduced plasma membrane localization, which effectively prevents TF from participating in budding or being incorporated into virus particles. Investigation of the regulation of TF will aid current efforts in the alphavirus field searching for approaches to mitigate alphaviral disease in humans. Copyright © 2017 American Society for Microbiology.

  4. INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

    Science.gov (United States)

    Grzegorzewicz, Anna E.; Pham, Ha; Gundi, Vijay A. K. B.; Scherman, Michael S.; North, Elton J.; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E. M.; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard E.; McNeil, Michael R.; Jackson, Mary

    2011-01-01

    New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. PMID:22344175

  5. Free-Flow Electrophoresis of Plasma Membrane Vesicles Enriched by Two-Phase Partitioning Enhances the Quality of the Proteome from Arabidopsis Seedlings.

    Science.gov (United States)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet T; Meents, Miranda J; Lao, Jeemeng; González Fernández-Niño, Susana M; Petzold, Christopher J; Frommer, Wolf B; Samuels, A Lacey; Heazlewood, Joshua L

    2016-03-04

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

  6. Differential effects of inhibitors and detergents on the Ca2+-ATPase and Mg2+-ATPase activities of the plasma membrane of a human oat cell carcinoma

    International Nuclear Information System (INIS)

    Knowles, A.F.; Lawrence, C.M.

    1986-01-01

    Plasma membranes of human oat cell carcinoma possess Mg 2+ - and Ca 2+ -dependent ATPase activities of similar magnitude. These activities exhibit the unusual characteristic of being inactiviated by prolonged incubation of the membrane with 1-2 mM dithiothreitol (DTT). Inactivation by DTT was prevented by lowering the incubation temperature, elevation of the membrane protein concentration, and addition of ATP. Fluorosulfonylbenzoyl adenosine (FSBA), an affinity ATP analog, also inactivates these activities. The Ca 2+ -ATPase activity appears to be more sensitive to both DTT and FSBA. The Ca 2+ -ATPase activity is more easily inactivated by Triton X-100, while the Mg 2+ -ATPase is preferentially activated by digitonin. These differential effects of inhibitors and detergents suggest that the Ca 2+ -ATPase and Mg 2+ -ATPase are separate enzymes. Incubation of oat cell carcinoma plasma membrane with [ 3 H]FSBA resulted in the labeling of several proteins. A labelled 35,000 dalton protein corresponds to the molecular weight of the oat cell carcinoma plasma membrane Ca 2+ -ATPase previously purified in this laboratory. The identity of one or more of the other labelled proteins with the Mg 2+ -ATPase has not been demonstrated, but is presently under investigation

  7. The Chemical Potential of Plasma Membrane Cholesterol: Implications for Cell Biology.

    Science.gov (United States)

    Ayuyan, Artem G; Cohen, Fredric S

    2018-02-27

    Cholesterol is abundant in plasma membranes and exhibits a variety of interactions throughout the membrane. Chemical potential accounts for thermodynamic consequences of molecular interactions, and quantifies the effective concentration (i.e., activity) of any substance participating in a process. We have developed, to our knowledge, the first method to measure cholesterol chemical potential in plasma membranes. This was accomplished by complexing methyl-β-cyclodextrin with cholesterol in an aqueous solution and equilibrating it with an organic solvent containing dissolved cholesterol. The chemical potential of cholesterol was thereby equalized in the two phases. Because cholesterol is dilute in the organic phase, here activity and concentration were equivalent. This equivalence allowed the amount of cholesterol bound to methyl-β-cyclodextrin to be converted to cholesterol chemical potential. Our method was used to determine the chemical potential of cholesterol in erythrocytes and in plasma membranes of nucleated cells in culture. For erythrocytes, the chemical potential did not vary when the concentration was below a critical value. Above this value, the chemical potential progressively increased with concentration. We used standard cancer lines to characterize cholesterol chemical potential in plasma membranes of nucleated cells. This chemical potential was significantly greater for highly metastatic breast cancer cells than for nonmetastatic breast cancer cells. Chemical potential depended on density of the cancer cells. A method to alter and fix the cholesterol chemical potential to any value (i.e., a cholesterol chemical potential clamp) was also developed. Cholesterol content did not change when cells were clamped for 24-48 h. It was found that the level of activation of the transcription factor STAT3 increased with increasing cholesterol chemical potential. The cholesterol chemical potential may regulate signaling pathways. Copyright © 2018. Published by

  8. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  9. Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

    Science.gov (United States)

    Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

    2015-04-01

    Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

  10. Cell cycle dependent changes in the plasma membrane organization of mammalian cells.

    Science.gov (United States)

    Denz, Manuela; Chiantia, Salvatore; Herrmann, Andreas; Mueller, Peter; Korte, Thomas; Schwarzer, Roland

    2017-03-01

    Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Studies on the postnatal development of the rat liver plasma membrane following maternal ethanol ingestion

    Energy Technology Data Exchange (ETDEWEB)

    Rovinski, B

    1984-01-01

    Studies on the developing rat liver and on the structure and function of the postnatal rat liver plasma membrane were carried out following maternal consumption of alcohol during pregnancy and lactation. A developmental study of alcohol dehydrogenase (ADH) indicated that both the activity and certain kinetic properties of the enzyme from the progeny of alcohol-fed and pair-fed mothers were similar. Fatty liver, however, developed in the alcoholic progeny only after ADH appeared on a day 19 of gestation. Further studies on structural and functional changes were then undertaken on the postnatal development of the rat liver plasma membrane. Radioligand binding studies performed using the hapatic alpha{sub 1}-adrenergic receptor as a plasma membrane probe demonstrated a significant decrease in receptor density in the alcoholic progeny, but no changes in binding affinity. Finally, the fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined. All these observations suggest that membrane alterations in the newborn may be partially responsible for the deleterious action(s) of maternal alcoholism at the molecular level.

  12. Luteinizing hormone-releasing hormone inactivation by purified pituitary plasma membranes: effects of receptor-binding studies.

    Science.gov (United States)

    Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C

    1979-05-01

    Inactivation of LHRH by purified bovine pituitary plasma membranes was studied in vitro. After incubation of [125I]iodo-LHRH with plasma membranes, the amount of tracer bound to the pellet was measured, and the integrity of the unbound tracer in the supernatant was assessed. Reduction in ability to bind to anti-LHRH serum and to rebind to plasma membranes together with altered electrophoretic mobility on polyacrylamide gels showed that the unbound [125I]iodo-LHRH was inactivated. LHRH inactivation occurred rapidly and was dependent upon membrane concentration and incubation temperature. These results indicate that hormone inactivation must be taken into account in the interpretation of LHRH-receptor interactions. During 37 C incubations, the apparent absence of specific LHRH binding can be explained by inactivation of tracer hormone. Significant LHRH inactivation also occurred at 0 C, which in part explains the insensitivity of LHRH receptor assays. Assessment of LHRH inactivation by different particulate subcellular fractions of pituitary tissue showed that the inactivating enzyme was associated with the plasma membranes; other organelles did not alter LHRH. The enzyme appeared to be an integral part of the plasma membrane structure, since enzymic activity could not be removed by washing without reducing specific LHRH binding. Additionally, reduction of LHRH inactivation by the inhibitors Bacitracin and Trasylol and by magnesium was also accompanied by reduced LHRH binding. Previous studies have shown that the majority of LHRH binding to pituitary plasma membranes is to the low affinity site (approximately 10(-6) M), but the significance of this binding has been uncertain. Our findings indicate that low affinity binding probably represents binding of LHRH to the inactivating enzyme. The LHRH analog, D-Ser6(TBu), des Gly10, ethylamide, has greater biological activity than LHRH and is not inactivated to a significant extent by pituitary plasma membranes. The

  13. Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

    Science.gov (United States)

    Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

    2017-07-05

    Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

    International Nuclear Information System (INIS)

    Dominguez, J.M.; Lanoix, J.; Paiement, J.

    1991-01-01

    We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha- 32 P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha- 32 P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations

  15. Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

    Science.gov (United States)

    Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

    2000-04-01

    Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

  16. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

    Science.gov (United States)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  17. Lateral mobility of plasma membrane lipids in a molluscan egg: Evidence for an animal/vegetal polarity

    OpenAIRE

    Laat, S.W. de; Speksnijder, J.E.; Dohmen, M.R.; Zoelen, E. van; Tertoolen, L.G.J.; Bluemink, J.G.

    1984-01-01

    The lateral diffusion of the lipid analog C₁₄-diI (3', 3'-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from fertilized egg up to 8-cell stage) the diffusion coefficient (D) of the mobile fraction (MF) of C₁₄-diI is significantly higher in the plasma membrane of the polar lobe as compared to the plasma membrane of the anima...

  18. Structure and electrochemical properties of the track membranes modified by tetrafluoroethane plasma

    International Nuclear Information System (INIS)

    Kravets, L.I.; Dmitriev, S.N.; Goryacheva, T.A.; Satulu, V.; Mitu, B.; Dinescu, G.

    2010-01-01

    A structure and charge transport properties of the poly(ethylene terephthalate) track membrane modified by the 1,1,1,2-tetrafluoroethane plasma have been studied. It has been found that the polymer deposition on the surface of a track membrane via the plasma polymerization of 1,1,1,2-tetrafluoroethane results in the creation of bilayered composite membranes that possess a conductivity asymmetry in electrolyte solutions - a rectification effect similar to that of p-n junction in semiconductors. This effect is caused by an important reduction of the pore diameter in the polymer layer that leads to changing the pore geometry as well as by existence of an interface between two layers with different concentrations of carboxyl groups. Information about the charge transport in the studied membranes has been obtained by the method of impedance spectroscopy

  19. Biochemical characterization of the plasma membrane H+ - ATPase from red beet (Beta vulgaris) hypocotyl tissue

    International Nuclear Information System (INIS)

    Oleski, N.A.

    1986-01-01

    Several biochemical techniques including selective solubilization followed by gel filtration or various types of affinity chromatography, and antibody production were employed in an attempt to purify the plasma membrane H + - ATPase from red beet hypocotyl tissue. While the enzyme could not be purified using any of these methods, it was possible to successfully conduct a more detailed biochemical analysis of the H + - ATPase. The molecular weight and isoelectric point of the enzyme were determined using N,N'dicyclohexylcarbodiimide (DCCD) and a H + - ATPase antibody. When plasma membrane vesicles were incubated with 20 μM [ 14 C]-DCCD at 0 C, a single 97,000 dalton protein was apparent on a fluorograph. A close correlation between [ 14 C]-DCCD labelling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentrations suggests that this 97,000 dalton protein is a component of the plasma membrane H + - ATPase. An antibody raised against the plasma membrane H + - ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the enzyme to be pH 6.5

  20. The influence of blood plasma of irradiated animals on activity of Ca2+ - ATPase and Mg2+ - ATPase in plasma membrane of thymocytes

    International Nuclear Information System (INIS)

    Dreval', V.I.

    1994-01-01

    Rats were irradiated at doses 1.5, 4.0, 7.0 and 10 Gy. After 1, 8, 15, 22 and 30 days the effect of blood plasma on activity of Ca 2+ -ATPase and Mg 2+ -ATPase in plasma membrane of thymocytes was investigated. It was found that the raise of irradiation dose leads to increasing of blood plasma effect on membrane-bound enzymes

  1. Modification of the poly(ethylene) terephthalate track membrane structure and surface in the plasma of non-polymerized gases

    International Nuclear Information System (INIS)

    Kravets, L.I.; Dmitriev, S.N.; Apel, P.Y.

    1999-01-01

    An investigation of the properties of poly(ethylene) terephthalate track membranes (PETTMs) treated with a plasma RF-discharge in non-polymerized gases has been performed. The influence of the plasma treatment conditions on the basic properties of the membranes has been studied. It was arranged that the effect of non-polymerized gases plasma on the PETTMs results to etching a membrane's surface layer. The membranes' pore size and the form in this case change. It is shown that it is possible to change the structure of track membranes directly by gas discharge etching

  2. SEM observations of particle track membrane surfaces modificated using plasma treatment

    International Nuclear Information System (INIS)

    Sartowska, B.; Buczkowski, M.; Starosta, W.

    2003-01-01

    This work presents results of scanning electron microscopy (SEM) observations of 0.4 μm membranes after plasma treatment with different parameters. The morphology changes at the surfaces and at the pore walls were observed. The character of changes in the membrane parameters according to the process conditions was determined

  3. Influence of quantum dot labels on single molecule movement in the plasma membrane

    DEFF Research Database (Denmark)

    Clausen, Mathias P.; Lagerholm, B. Christoffer

    2011-01-01

    Single particle tracking results are very dependent on the probe that is used. In this study we have investigated the influence that functionalized quantum dots (QDs) have on the recorded movement in single molecule tracking experiments of plasma membrane species in live cells. Potential issues...... for simultaneous investigations of different plasma membrane species in order to discriminate the effect of the label from differences in movement of the target molecules....

  4. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase

    DEFF Research Database (Denmark)

    Jahn, T.; Fuglsang, A.T.; Olsson, A.

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H...... plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase....

  5. Surface Modification of Asymmetric Polysulfone/Polyethylene Glycol Membranes by DC Ar-Glow Discharge Plasma

    Directory of Open Access Journals (Sweden)

    Chalad Yuenyao

    2016-01-01

    Full Text Available Polysulfone/polyethylene glycol (PSF/PEG membranes were prepared by dry/wet phase inversion method. Effects of direct current glow discharge plasma using argon as working gas on morphological structures and gas separation properties of membranes were studied. Alteration of membrane characteristics were analyzed by various techniques like contact angle, scanning electron microscope, Fourier transform infrared spectroscopy, and dynamic mechanical thermal analysis. Gas separation properties were measured in terms of permeation and ideal O2/N2 selectivity. Results showed that hydrophilic and gas separation properties of PSF/PEG membranes increased by plasma surface modification. It was also shown that the dosage of PEG and plasma treatment affected the morphological structures and mechanical and gas separation properties. The macro voids and transmembrane structure disappeared with a little amount of PEG dosage. Pore size and mechanical strength tend to decrease with increasing PEG dosage up to 10 wt%. Glass transition temperature (Tg receded from 201.8 to 143.7°C for pure PSF and PSF/PEG with PEG dosage of 10 wt%. O2 and N2 gases permeation through the 10-minute plasma treated membranes tend to increase. However, the permeation strongly dispersed when treatment time was more extended.

  6. Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery

    Energy Technology Data Exchange (ETDEWEB)

    Kurkdjian, A.; Leitz, G.; Manigault, P.; Harim, A.; Greulich, K. O.

    1993-07-01

    Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few μm in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The movement of the protoplasm is retarded by increases in calcium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain preferentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The complete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the presence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Conditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plasmolysis and laser microperforation. This laser technique should be suitable for the performance of electrophysiological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells. (author)

  7. Non-enzymatic access to the plasma membrane of Medicago root hairs by laser microsurgery

    International Nuclear Information System (INIS)

    Kurkdjian, A.; Leitz, G.; Manigault, P.; Harim, A.; Greulich, K.O.

    1993-01-01

    Using UV laser microsurgery, the cell walls of root hairs from Medicago sativa (alfalfa) were perforated under plasmolysing conditions, giving direct access to the plasma membrane without enzyme treatment. The opening in the cell wall of a few μm in diameter results in immediate movement of the protoplasm and partial or complete extrusion of the cell contents. The movement of the protoplasm is retarded by increases in calcium concentration. The calcium-dependency of the movement of the protoplasm allows us to obtain preferentially the extrusion of protoplasm, or to gain access to a small area of plasma membrane in situ. The complete protoplasm can be expelled, to form a protoplast. Fluorescein diacetate staining indicated esterase activity and membrane integrity of the protoplasts. Microscopic examination revealed organelle movement and the presence of a nucleus. The plasma membrane was free from cell wall fragments, as shown by Tinopal staining. Conditions for obtaining plasmolysis without disturbing the physiology of the root hairs too much were achieved by slow, stepwise and reversible plasmolysis. Cytoplasmic streaming in root hairs was maintained during plasmolysis and laser microperforation. This laser technique should be suitable for the performance of electrophysiological studies using the patch-clamp technique on plasma membrane from non-enzyme-treated cells. (author)

  8. A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains[S

    Science.gov (United States)

    Krager, Kimberly J.; Sarkar, Mitul; Twait, Erik C.; Lill, Nancy L.; Koland, John G.

    2012-01-01

    The submicroscopic spatial organization of cell surface receptors and plasma membrane signaling molecules is readily characterized by electron microscopy (EM) via immunogold labeling of plasma membrane sheets. Although various signaling molecules have been seen to segregate within plasma membrane microdomains, the biochemical identity of these microdomains and the factors affecting their formation are largely unknown. Lipid rafts are envisioned as submicron membrane subdomains of liquid ordered structure with differing lipid and protein constituents that define their specific varieties. To facilitate EM investigation of inner leaflet lipid rafts and the localization of membrane proteins therein, a unique genetically encoded reporter with the dually acylated raft-targeting motif of the Lck kinase was developed. This reporter, designated Lck-BAP-GFP, incorporates green fluorescent protein (GFP) and biotin acceptor peptide (BAP) modules, with the latter allowing its single-step labeling with streptavidin-gold. Lck-BAP-GFP was metabolically biotinylated in mammalian cells, distributed into low-density detergent-resistant membrane fractions, and was readily detected with avidin-based reagents. In EM images of plasma membrane sheets, the streptavidin-gold-labeled reporter was clustered in 20–50 nm microdomains, presumably representative of inner leaflet lipid rafts. The utility of the reporter was demonstrated in an investigation of the potential lipid raft localization of the epidermal growth factor receptor. PMID:22822037

  9. Characterization of phospholipid composition and its control in the plasma membrane of developing soybean root

    International Nuclear Information System (INIS)

    Whitman, C.E.

    1985-01-01

    The phospholipid composition of plasma membrane enriched fractions from developing soybean root and several mechanisms which may regulate it have been examined. Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots (Glycine max [L.] Merr. Cult. Wells II). Analysis of lipid extracts revealed two major phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Minor phospholipid classes were phosphatidylinositol, phosphatidylserine, phosphatidylgylcerol and diphosphatidylgylcerol. Phospholipid composition was similar at each developmental stage. Fatty acids of phosphatidylcholine and phosphatidylethanolamine were 16:0, 18:0, 18:2, and 18:3. Fatty acid composition varied with both phospholipid class and the developmental stage of the root. The degradation of phosphatidylcholine by endogenous phospholipase D during membrane isolation indicated that this enzyme might be involved in phospholipid turnover within the membrane. Phospholipase D activity was heat labile and increasing the pH of the enzyme assay from 5.3 to 7.8 resulted in 90% inhibition of activity. The turnover of fatty acids within the phospholipids of the plasma membrane was studied. Mature root sections were incubated with [1- 14 C] acetate, 1 mM Na acetate and 50 μg/ml chloramphenicol. Membrane lipid extracts analyzed for phospholipid class and acyl chain composition revealed that the long incubation times did not alter the phospholipid composition of the plasma membrane enriched fraction

  10. Modulation of Erythrocyte Plasma Membrane Redox System Activity by Curcumin

    Directory of Open Access Journals (Sweden)

    Prabhakar Singh

    2016-01-01

    Full Text Available Plasma membrane redox system (PMRS is an electron transport chain system ubiquitously present throughout all cell types. It transfers electron from intracellular substrates to extracellular acceptors for regulation of redox status. Curcumin, isolated from Curcuma longa, has modulatory effects on cellular physiology due to its membrane interaction ability and antioxidant potential. The present study investigates the effect of curcumin on PMRS activity of erythrocytes isolated from Wistar rats in vitro and in vivo and validated through an in silico docking simulation study using Molegro Virtual Docker (MVD. Effects of curcumin were also evaluated on level of glutathione (GSH and the oxidant potential of plasma measured in terms of plasma ferric equivalent oxidative potentials (PFEOP. Results show that curcumin significantly (p<0.01 downregulated the PMRS activity in a dose-dependent manner. Molecular docking results suggest that curcumin interacts with amino acids at the active site cavity of cytochrome b5 reductase, a key constituent of PMRS. Curcumin also increased the GSH level in erythrocytes and plasma while simultaneously decreasing the oxidant potential (PFEOP of plasma. Altered PMRS activity and redox status are associated with the pathophysiology of several health complications including aging and diabetes; hence, the above finding may explain part of the role of curcumin in health beneficial effects.

  11. The effect of ultraviolet radiation on wheat root vesicles enriched in plasma membrane

    International Nuclear Information System (INIS)

    Wright, L.A. Jr.; Murphy, T.M.; Travis, R.L.

    1981-01-01

    The irradiation of plant cells with UV radiation (254 nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K + -stimulated ATPase was very sensitive to UV radiation (100% inhibition with 2 ). ATPase activity measured in the absence of K + and K + -stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m 2 . (author)

  12. The dynamic interplay of plasma membrane domains and cortical microtubules in secondary cell wall patterning

    Directory of Open Access Journals (Sweden)

    Yoshihisa eOda

    2013-12-01

    Full Text Available Patterning of the cellulosic cell wall underlies the shape and function of plant cells. The cortical microtubule array plays a central role in the regulation of cell wall patterns. However, the regulatory mechanisms by which secondary cell wall patterns are established through cortical microtubules remain to be fully determined. Our recent study in xylem vessel cells revealed that a mutual inhibitory interaction between cortical microtubules and distinct plasma membrane domains leads to distinctive patterning in secondary cell walls. Our research revealed that the recycling of active and inactive ROP proteins by a specific GAP and GEF pair establishes distinct de novo plasma membrane domains. Active ROP recruits a plant-specific microtubule-associated protein, MIDD1, which mediates the mutual interaction between cortical microtubules and plasma membrane domains. In this mini review, we summarize recent research regarding secondary wall patterning, with a focus on the emerging interplay between plasma membrane domains and cortical microtubules through MIDD1 and ROP.

  13. Correlation between Balmer α emission and hydrogen flux through a superpermeable niobium membrane in a low-pressure multicusp plasma source

    International Nuclear Information System (INIS)

    Bruneteau, A.M.; Notkin, M.E.; Livshits, A.I.; Bacal, M.

    2002-01-01

    The purpose of this paper is to correlate hydrogen or deuterium flux through super permeable membranes with incident hydrogen or deuterium atom flux from the plasma. To this aim a hydrogen or deuterium plasma is created in a hybrid multicusp plasma source. We investigate Balmer α emission from the multicusp plasma and the output pressure behind a superpermeable niobium membrane immersed in the plasma.The output pressure is proportional to the flux of atoms and ions arriving on the membrane. We find that both output pressure and excited atoms emission satisfy plasma parameters relations. It is thus verified that plasma-driven superpermeation of hydrogen is due essentially to neutral atoms from the plasma incident to the membrane

  14. Characterization of the cytochrome c oxidase in isolated and purified plasma membranes from the cyanobacterium Anacystis nidulans

    International Nuclear Information System (INIS)

    Peschek, G.A.; Wastyn, M.; Trnka, M.; Molitor, V.; Fry, I.V.; Packer, L.

    1989-01-01

    Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [ 35 S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min -1 (mg of protein) -1 , respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa 3 -type enzyme from the properties of its redox-active and EDTA-resistant Cu 2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa 3 -type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa 3 -type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme

  15. Leucine-based receptor sorting motifs are dependent on the spacing relative to the plasma membrane

    DEFF Research Database (Denmark)

    Geisler, C; Dietrich, J; Nielsen, B L

    1998-01-01

    Many integral membrane proteins contain leucine-based motifs within their cytoplasmic domains that mediate internalization and intracellular sorting. Two types of leucine-based motifs have been identified. One type is dependent on phosphorylation, whereas the other type, which includes an acidic...... amino acid, is constitutively active. In this study, we have investigated how the spacing relative to the plasma membrane affects the function of both types of leucine-based motifs. For phosphorylation-dependent leucine-based motifs, a minimal spacing of 7 residues between the plasma membrane...... and the phospho-acceptor was required for phosphorylation and thereby activation of the motifs. For constitutively active leucine-based motifs, a minimal spacing of 6 residues between the plasma membrane and the acidic residue was required for optimal activity of the motifs. In addition, we found that the acidic...

  16. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  17. Isolation of plasma membranes from the nervous system by countercurrent distribution in aqueous polymer two-phase systems.

    Science.gov (United States)

    Schindler, Jens; Nothwang, Hans Gerd

    2009-01-01

    The plasma membrane separates the cell-interior from the cell's environment. To maintain homeostatic conditions and to enable transfer of information, the plasma membrane is equipped with a variety of different proteins such as transporters, channels, and receptors. The kind and number of plasma membrane proteins are a characteristic of each cell type. Owing to their location, plasma membrane proteins also represent a plethora of drug targets. Their importance has entailed many studies aiming at their proteomic identification and characterization. Therefore, protocols are required that enable their purification in high purity and quantity. Here, we report a protocol, based on aqueous polymer two-phase systems, which fulfils these demands. Furthermore, the protocol is time-saving and protects protein structure and function.

  18. Free-flow electrophoresis of plasma membrane vesicles enriched by two-phase partitioning enhances the quality of the proteome from Arabidopsis seedlings

    DEFF Research Database (Denmark)

    de Michele, Roberto; McFarlane, Heather E; Parsons, Harriet Tempé

    2016-01-01

    The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane...... using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane...... isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including...

  19. Plasma membrane calcium ATPases and related disorders.

    Science.gov (United States)

    Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

    2013-03-01

    The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Molecular size estimation of plasma membrane β-glucan synthase from red beet root

    International Nuclear Information System (INIS)

    Sloan, M.E.; Eiberger, L.L.; Wasserman, B.P.

    1986-01-01

    Cellulose and cell wall β-D-glucans in higher plants are thought to be synthesized by the plasma membrane enzyme, β-glucan synthase. This enzyme has never been purified to homogeneity, hence its subunit composition is unknown. Partial purification of red beet root glucan synthase by glycerol density gradient centrifugation followed by SDS-PAGE yielded a highly enriched subunit of 68 kDa. Radiation inactivation of plasma membranes gave a molecular size the 450 kDa for the holoenzyme complex. This suggests that glucan synthase consists of 6 to 7 subunits and confirms electron microscope studies showing that glucan synthases exist as multi-subunit complexes embedded within the membrane

  1. Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

    Science.gov (United States)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-02-05

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

  2. Steric exclusion and protein conformation determine the localization of plasma membrane transporters

    NARCIS (Netherlands)

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

  3. Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential

    NARCIS (Netherlands)

    Duijn, Bert van; Vogelzang, Sake A.; Ypey, Dirk L.; Molen, Loek G. van der; Haastert, Peter J.M. van

    1990-01-01

    We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be

  4. Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo.

    Science.gov (United States)

    Speksnijder, J E; Dohmen, M R; Tertoolen, L G; de Laat, S W

    1985-07-01

    Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.

  5. Tailored adhesion behavior of polyelectrolyte thin films deposited on plasma-treated poly(dimethylsiloxane) for functionalized membranes

    Energy Technology Data Exchange (ETDEWEB)

    Bassil, Joelle, E-mail: joelle.bassil@univ-lorraine.fr [Institut Jean Lamour (IJL), UMR CNRS 7198, Université de Lorraine, Parc de Saurupt CS50840, 54011 Nancy (France); Alem, Halima, E-mail: halima.alem@univ-lorraine.fr [Institut Jean Lamour (IJL), UMR CNRS 7198, Université de Lorraine, Parc de Saurupt CS50840, 54011 Nancy (France); Henrion, Gérard, E-mail: gerard.henrion@univ-lorraine.fr [Institut Jean Lamour (IJL), UMR CNRS 7198, Université de Lorraine, Parc de Saurupt CS50840, 54011 Nancy (France); Roizard, Denis, E-mail: denis.roizard@univ-lorraine.fr [Laboratoire Réactions et Génie des Procédés (LRGP), UMR CNRS 7274, ENSIC, Université de Lorraine, 1 rue Grandville, 54011 Nancy (France)

    2016-04-30

    Graphical abstract: - Highlights: • The surface of PDMS membrane was first modified by Ar/O{sub 2} plasma to increase its surface energy. • Subsequently, a homogeneous multilayer of the well-known couple of polyelectrolyte PDADMAC/PSS was deposited on the plasma treated PDMS. • The relation between the parameters of the modification processes and the morphology, wettability, structure and adhesion of the polyelectrolytes layers based PDMS membranes is investigated and enlightened. - Abstract: Completely homogenous films formed via the layer-by-layer assembly of poly(diallyldimethylammonium chloride) (PDADMAC) and the poly(styrene sulfonate) were successfully obtained on plasma-treated poly(dimethylsiloxane) (PDMS) substrates. To modify the hydrophobicity of the PDMS surface, a cold plasma treatment was previously applied to the membrane, which led to the creation of hydrophilic groups on the surface of the membrane. PDMS wettability and surface morphology were successfully correlated with the plasma parameters. A combination of contact angle measurements, scanning electron microscopy (SEM) and atomic force microscopy (AFM) analysis was used to demonstrate that homogeneous and hydrophilic surfaces could be achieved on PDMS cold-plasma-treated membranes. The stability of the assembled PEL layer on the PDMS was evaluated using a combination of pull-off testing and X-ray photoelectron spectroscopy (XPS), which confirmed the relevance of a plasma pre-treatment as the adhesion of the polyelectrolyte multilayers was greatly enhanced when the deposition was completed on an activated PDMS surface at 80 W for 5 min.

  6. Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

    Science.gov (United States)

    Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

    2008-01-01

    Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

  7. RF plasma-driven hydrogen permeation through a biased iron membrane

    International Nuclear Information System (INIS)

    Banno, T.; Waelbroeck, F.; Winter, J.

    1984-01-01

    The steady-state RF plasma-driven hydrogen permeation through an electrically biased iron membrane has been investigated as a function of the bias potential Vsub(M) for membrane temperatures in the range of 150-400 0 C. Vsub(M) has been gradually increased positively from the floating potential of the membrane. The permeation flux decreases when Vsub(M) increases at low voltages: positive hydrogen ions are repelled. The membrane temperature does not influence this effect measurably. The permeation flux starts to increase when Vsub(M) is raised higher, i.e. when energetic electrons strike the surface. This phenomenon shows a pronounced temperature dependence - the enhancement is largest for the lowest temperatures. The effect is interpreted in terms of an electron-induced dissociation of hydrogen molecules on the membrane surface. (orig.)

  8. Characterization of plant plasma membrane antigens: [Annual] progress report

    International Nuclear Information System (INIS)

    Galbraith, D.W.; Afonso, C.L.; Meyer, D.; Harkins, K.R.

    1987-01-01

    Protoplast plasma membranes were used to raise antibodies in mice to cell surface antigens. Monoclonal antibodies were selected from those produced and used for indirect immunofluorescence microscopic analysis of N. tabacum cells. In parallel studies cDNA expression libraries were prepared. (DT)

  9. Calcium fluxes across the plasma membrane of Commelina communis L. assayed in a cell-free system

    International Nuclear Information System (INIS)

    Siebers, B.; Graef, P.; Weiler, E.W.

    1990-01-01

    The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with 45 Ca 2+ through the action of the plasma membrane Ca 2+ -ATPase. Results suggest the presence of a Ca 2+ channel in the plasma membrane of C. communis. The channel is obtained in a Ca 2+ -inactivated state after preparation and Ca 2+ -loading of the vesicles. The inactivation is removed by TFP [trifluoperazine] or W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], presumably due to the Ca 2+ -mobilizing effect of these compounds. The activated Ca 2+ channel is La 3+ sensitive and, in the cell, would allow for passage of Ca 2+ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed

  10. Plasma lipid pattern and red cell membrane structure in β-thalassemia patients in Jakarta

    Directory of Open Access Journals (Sweden)

    Seruni K.U. Freisleben

    2011-08-01

    Full Text Available Background: Over the last 10 years, we have investigated thalassemia patients in Jakarta to obtain a comprehensive picture of iron overload, oxidative stress, and cell damage.Methods: In blood samples from 15 transfusion-dependent patients (group T, 5 non-transfused patients (group N and 10 controls (group C, plasma lipids and lipoproteins, lipid-soluble vitamin E, malondialdehyde (MDA and thiol status were measured. Isolated eryhtrocyte membranes were investigated with electron paramagnetic resonance (EPR spectroscopy using doxyl-stearic acid and maleimido-proxyl spin lables. Data were analyzed statistically with ANOVA.Results: Plasma triglycerides were higher and cholesterol levels were lower in thalassemic patients compared to controls. Vitamin E, group C: 21.8 vs T: 6.2 μmol/L and reactive thiols (C: 144 vs. T: 61 μmol/L were considerably lower in transfused patients, who exert clear signs of oxidative stress (MDA, C: 1.96 vs T: 9.2 μmol/L and of tissue cell damage, i.e., high transaminases plasma levels. Non-transfused thalassemia patients have slight signs of oxidative stress, but no significant indication of cell damage. Erythrocyte membrane parameters from EPR spectroscopy differ considerably between all groups. In transfusion-dependent patients the structure of the erythrocyte membrane and the gradients of polarity and fluidity are destroyed in lipid domains; binding capacity of protein thiols in the membrane is lower and immobilized.Conclusion: In tranfusion-dependent thalassemic patients, plasma lipid pattern and oxidative stress are associated with structural damage of isolated erythrocyte membranes as measured by EPR spectroscopy with lipid and proteinthiol spin labels. (Med J Indones 2011; 20:178-84Keywords: electron paramagnetic resonance spectroscopy, erythrocyte membrane, lipoproteins, oxidative stress, thalassemia, plasma lipids.

  11. Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

    Science.gov (United States)

    Golan, Yonatan; Sherman, Eilon

    2017-06-01

    The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

  12. Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS

    DEFF Research Database (Denmark)

    Schneider, Falk; Waithe, Dominic; Clausen, Mathias P

    2017-01-01

    (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live cell plasma membrane and in actin cytoskeleton-free cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids......Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling, and they are suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy...... forming immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules, and highlight a powerful experimental approach to decipher specific influences on molecular plasma...

  13. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw

    2014-01-01

    Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1 was developed to enable...... routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely...... to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...

  14. Characterization of a light-controlled anion channel in the plasma membrane of mesophyll cells of pea

    NARCIS (Netherlands)

    Elzenga, J.T.M.; Volkenburgh Van, E

    In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp

  15. Effect of saline stress on plasma membrane structure and function of barley roots

    International Nuclear Information System (INIS)

    Rahmani, F. H.

    2000-01-01

    Barely (Hordeum vulgare L. c v. Black Local) plants were grown hydroponic ally under different saline stresses (50, 100, 150 And 200 mm NaCI. The adverse effect of each saline stress on the structure and function of root cells plasma membrane was studied in terms of root surface ATPase activation by NaCI in the reaction mixture. Was 0, 50, 100. 150 and 200mM. ATPase activity was found to be increased gradually at certain concentrations of NaCI. For control and 50mM stressed plants, the increase in root surface ATPase activity was started at 150mM NaCI. For 100mM stressed plants it was started at 100mM NaCI. For 150 and 200mM stressed plants it was stated at 50mM NaCI Results indicated that the adverse effect of the growth medium saline stresses on the integrity of the plasma membrane was started at 100mM saline stress. Accordingly the role of plasma membrane bound ATPase in active ion transport was disturbed at 100mM saline stress and may be impaired at 150 and 200mM saline stresses. It was suggested that the lipid environment of the plasma membrane surrounding ATPase was modified by the saline stresses 100-200mM. (author). 38 refs., 2 figs., 2 tabs

  16. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  17. Review of low pressure plasma processing of proton exchange membrane fuel cell electrocatalysts

    OpenAIRE

    Brault , Pascal

    2016-01-01

    Review article; International audience; The present review is describing recent advances in plasma deposition and treatment of low temperature proton exchange membrane fuel cells electrocatalysts. Interest of plasma processing for growth of platinum based, non-precious and metal free electrocatalysts is highlighted. Electrocatalysts properties are tentatively correlated to plasma parameters.

  18. Identification of antifungal H+-ATPase inhibitors with effect on the plasma membrane potential

    DEFF Research Database (Denmark)

    Kjellerup, Lasse; Gordon, Sandra; Cohrt, Karen O'Hanlon

    2017-01-01

    to depolarize the membrane and inhibit extracellular acidification in intact fungal cells, concomitant with a significant increase in intracellular ATP levels. Collectively, we suggest these effects may be a common feature for Pma1 inhibitors. Additionally, the work uncovered a dual mechanism for the previously......The plasma membrane H(+)-ATPase (Pma1) is an essential fungal protein and a proposed target for new antifungal medications. A small-molecule library containing ∼191,000 commercially available compounds was screened for inhibition of S. cerevisiae plasma membranes containing Pma1. The overall hit...... identified cationic peptide BM2, revealing fungal membrane disruption in addition to Pma1 inhibition. The methods presented here provide a solid platform for the evaluation of Pma1-specific inhibitors in a drug development setting. The present inhibitors could serve as a starting point for the development...

  19. Characterization and quantitation of concanavalin A binding by plasma membrane enriched fractions from soybean root

    International Nuclear Information System (INIS)

    Berkowitz, R.L.; Travis, R.L.

    1981-01-01

    The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritated ( 3 H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of 3 H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10 15 molecules of 3 H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside. A major peak of 3 H-Con A binding was also observed in fractions recovered from the 25/30% interface of a complex discontinuous sucrose density gradient when membranes were isolated in the absence of Mg 2+ . When high Mg 2+ was present in the isolation and gradient media, the peak was shifted to a fraction recovered from the 34/38% sucrose interface. These results suggest that Con A binding sites are also present on membranes of the endoplasmic reticulum. The amount of Con A bound by endoplasmic reticulum membranes was at least twice the amount bound by membranes in plasma membrane enriched fractions when binding was compared on a per unit membrane protein basis. In contrast, mitochondrial inner membranes, which equilibrate at the same density as plasma membranes, had little ability to bind the lectin

  20. Ultrastructural modification of the plasma membrane in HUT 102 lymphoblasts by long-wave ultraviolet light, psoralen, and PUVA

    International Nuclear Information System (INIS)

    Malinin, G.I.; Lo, H.K.; Hornicek, F.J.; Malinin, T.I.

    1990-01-01

    Ultrastructural alterations of the plasma membrane in HUT 102 lymphoblasts were assessed after a 2-h interaction with a suprapharmacologic (15 micrograms/ml) concentration of 8-MOP, 2-h irradiation with UVA (2.1 mW/cm2), and the exposure of the HUT 102 cells to PUVA under the same conditions. The dark reaction of HUT cells with 8-MOP resulted in the disappearance of microvilli, the emergence of plasma-membrane-associated spherical bodies, formation of lamellar fungiform membrane evaginations, and, in approximately 1% of the cells, formation of uropods and cell capping. Except for uropod formation and cell capping, UVA has induced the same plasma-membrane alterations, and was more deleterious to structural cytoplasmic integrity than 8-MOP. Morphologic changes of the plasma membrane in PUVA-exposed cells tended to replicate structural alterations elicited independently during the dark reaction by suprapharmacologic 8-MOP concentrations. Partial retention of microvilli by cells after PUVA was the sole exception. In light of all available evidence we conclude that psoralen during the dark reactions interacts with plasma membrane lipids by as yet undisclosed mechanisms and that in addition to lipids, membrane proteins are also the primary target of the initial interaction of HUT 102 cells with psoralen during PUVA treatment

  1. The ascorbate carrier of higher plant plasma membranes preferentially translocates the fully oxidized (dehydroascorbate) molecule

    International Nuclear Information System (INIS)

    Horemans, N.; Asard, H.; Caubergs, R.J.

    1997-01-01

    Recently, the uptake of 14C-labeled ascorbate (ASC) into highly purified bean (Phaseolus vulgaris L.) plasma membrane vesicles was demonstrated in our laboratory. However, the question of the redox status of the transported molecule (ASC or dehydroascorbate [DHA]) remained unanswered. In this paper we present evidence that DHA is transported through the plasma membrane. High-performance liquid chromatography analysis of the redox status of ASC demonstrated that freshly purified plasma membranes exhibit a high ASC oxidation activity. Although it is not yet clear whether this activity is enzymatic it complicates the interpretation of ASC-transport experiments in vitro and in vivo. In an attempt to correlate the ASC redox status to transport of the molecule, the ability of different compounds to reduce DHA was analyzed and their effect on ASC-transport activity tested. Administering of various reductants resulted in different levels of inhibition of ASC uptake (dithiothreitol dithioerythritol beta-mercaptoethanol beta-mercaptopropanol). Glutathione, cysteine, dithionite, and thiourea did not significantly affect ASC transport. Statistical analysis indicated a strong correlation of the Spearman rank correlation coefficient (Rs) of 0.919 (P = 0.0005, n = 9) between the level of ASC oxidation and the amount of transported molecules into the vesicles. The administering of ASC oxidants such as ferricyanide and ASC oxidase resulted in a stimulated ASC uptake into the plasma membrane vesicles. Together, our results demonstrate that a vitamin C carrier in purified bean plasma membranes translocates DHA from the apoplast to the cytosol

  2. Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

    Science.gov (United States)

    Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

    2004-07-01

    The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

  3. Mammalian gamete plasma membranes re-assessments and reproductive implications

    Science.gov (United States)

    Establishment of the diploid status occurs with the fusion of female and male gametes. Both the mammalian oocyte and spermatozoa are haploid cells surrounded with plasma membranes that are rich in various proteins playing a crucial role during fertilization. Fertilization is a complex and ordered st...

  4. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

    Science.gov (United States)

    Csermely, P; Szamel, M; Resch, K; Somogyi, J

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

  5. Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

    Science.gov (United States)

    Lu, Stella M; Fairn, Gregory D

    2018-04-01

    The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

  6. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

    Science.gov (United States)

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

  7. The product of the ABC half-transporter gene ABCG2 (BCRP/MXR/ABCP) is expressed in the plasma membrane

    DEFF Research Database (Denmark)

    Rocchi, E; Khodjakov, A; Volk, E L

    2000-01-01

    by Western blot and immunohistochemistry. This protein is highly overexpressed in several drug-resistant cell lines and localizes predominantly to the plasma membrane, instead of to intracellular membranes as seen with all other known half-transporters. Therefore, BCRP/MXR is unique among the ABC half......The products of the ABC gene family can be generally classified as either full-transporters of half-transporters. Full-transporters are expressed in the plasma membrane, whereas half-transporters are usually found in intracellular membranes. Recently, an ABC half-transporter, the ABCG2 gene product......-transporters by being localized to the plasma membrane....

  8. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Muratore, Massimo, E-mail: M.Muratore@ed.ac.uk [Institute of Integrated Micro and Nano System, School of Engineering, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Mitchell, Steve [Institute of Molecular Plant Science, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JF (United Kingdom); Waterfall, Martin [Institute of Immunology and Infection Research, School of Biological Science, The University of Edinburgh, Edinburgh EH9 3JT (United Kingdom)

    2013-09-06

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy.

  9. Plasma membrane characterization, by scanning electron microscopy, of multipotent myoblasts-derived populations sorted using dielectrophoresis

    International Nuclear Information System (INIS)

    Muratore, Massimo; Mitchell, Steve; Waterfall, Martin

    2013-01-01

    Highlights: •Dielectrophoretic separation/sorting of multipotent cells. •Plasma membrane microvilli structure of C2C12 and fibroblasts by SEM microscopy. •Cell cycle determination by Ki-67 in DEP-sorted cells. •Plasma membrane differences responsible for changes in membrane capacitance. -- Abstract: Multipotent progenitor cells have shown promise for use in biomedical applications and regenerative medicine. The implementation of such cells for clinical application requires a synchronized, phenotypically and/or genotypically, homogenous cell population. Here we have demonstrated the implementation of a biological tag-free dielectrophoretic device used for discrimination of multipotent myoblastic C2C12 model. The multipotent capabilities in differentiation, for these cells, diminishes with higher passage number, so for cultures above 70 passages only a small percentage of cells is able to differentiate into terminal myotubes. In this work we demonstrated that we could recover, above 96% purity, specific cell types from a mixed population of cells at high passage number without any biological tag using dielectrophoresis. The purity of the samples was confirmed by cytometric analysis using the cell specific marker embryonic myosin. To further investigate the dielectric properties of the cell plasma membrane we co-culture C2C12 with similar size, when in suspension, GFP-positive fibroblast as feeder layer. The level of separation between the cell types was above 98% purity which was confirmed by flow cytometry. These levels of separation are assumed to account for cell size and for the plasma membrane morphological differences between C2C12 and fibroblast unrelated to the stages of the cell cycle which was assessed by immunofluorescence staining. Plasma membrane conformational differences were further confirmed by scanning electron microscopy

  10. Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.

    Science.gov (United States)

    Hachez, Charles; Laloux, Timothée; Reinhardt, Hagen; Cavez, Damien; Degand, Hervé; Grefen, Christopher; De Rycke, Riet; Inzé, Dirk; Blatt, Michael R; Russinova, Eugenia; Chaumont, François

    2014-07-01

    Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane. © 2014 American Society of Plant Biologists. All rights reserved.

  11. Manipulation of host membranes by bacterial effectors.

    Science.gov (United States)

    Ham, Hyeilin; Sreelatha, Anju; Orth, Kim

    2011-07-18

    Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

  12. Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

    DEFF Research Database (Denmark)

    Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

    2012-01-01

    Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric...... of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase...... analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any...

  13. Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

    DEFF Research Database (Denmark)

    Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

    2005-01-01

    In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

  14. Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

    Science.gov (United States)

    Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

    2018-01-01

    Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

  15. Role of plasma membrane surface charges in dictating the feasibility of membrane-nanoparticle interactions

    Science.gov (United States)

    Sinha, Shayandev; Jing, Haoyuan; Sachar, Harnoor Singh; Das, Siddhartha

    2017-12-01

    Receptor-ligand (R-L) binding mediated interactions between the plasma membrane (PM) and a nanoparticle (NP) require the ligand-functionalized NPs to come to a distance of separation (DOS) of at least dRL (length of the R-L complex) from the receptor-bearing membranes. In this letter, we establish that the membrane surface charges and the surrounding ionic environment dictate whether or not the attainment of such a critical DOS is possible. The negatively charged membrane invariably induces a negative electrostatic potential at the NP surface, repelling the NP from the membrane. This is countered by the attractive influences of the thermal fluctuations and van der Waals (vdw) interactions that drive the NP close to the membrane. For a NP approaching the membrane from a distance, the ratio of the repulsive (electrostatic) and attractive (thermal and vdW) effects balances at a critical NP-membrane DOS of dg,c. For a given set of parameters, there can be two possible values of dg,c, namely, dg,c,1 and dg,c,2 with dg,c,1 ≫ dg,c,2. We establish that any R-L mediated NP-membrane interaction is possible only if dRL > dg,c,1. Therefore, our study proposes a design criterion for engineering ligands for a NP that will ensure the appropriate length of the R-L complex in order to ensure the successful membrane-NP interaction in the presence of a given electrostatic environment. Finally, we discuss the manner in which our theory can help designing ligand-grafted NPs for targeted drug delivery, design biomimetics NPs, and also explain various experimental results.

  16. Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

    Science.gov (United States)

    Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

    2002-05-01

    White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

  17. Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

    OpenAIRE

    Orsini, F.; Santacroce, M.; Cremona, A.; Gosvami, N. N.; Lascialfari, A.; Hoogenboom, B. W.

    2014-01-01

    Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23...

  18. Hemocompatible control of sulfobetaine-grafted polypropylene fibrous membranes in human whole blood via plasma-induced surface zwitterionization.

    Science.gov (United States)

    Chen, Sheng-Han; Chang, Yung; Lee, Kueir-Rarn; Wei, Ta-Chin; Higuchi, Akon; Ho, Feng-Ming; Tsou, Chia-Chun; Ho, Hsin-Tsung; Lai, Juin-Yih

    2012-12-21

    In this work, the hemocompatibility of zwitterionic polypropylene (PP) fibrous membranes with varying grafting coverage of poly(sulfobetaine methacrylate) (PSBMA) via plasma-induced surface polymerization was studied. Charge neutrality of PSBMA-grafted layers on PP membrane surfaces was controlled by the low-pressure and atmospheric plasma treatment in this study. The effects of grafting composition, surface hydrophilicity, and hydration capability on blood compatibility of the membranes were determined. Protein adsorption onto the different PSBMA-grafted PP membranes from human fibrinogen solutions was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Blood platelet adhesion and plasma clotting time measurements from a recalcified platelet-rich plasma solution were used to determine if platelet activation depends on the charge bias of the grafted PSBMA layer. The charge bias of PSBMA layer deviated from the electrical balance of positively and negatively charged moieties can be well-controlled via atmospheric plasma-induced interfacial zwitterionization and was further tested with human whole blood. The optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and keeps its original blood-inert property of antifouling, anticoagulant, and antithrmbogenic activities when it comes into contact with human blood. This work suggests that the hemocompatible nature of grafted PSBMA polymers by controlling grafting quality via atmospheric plasma treatment gives a great potential in the surface zwitterionization of hydrophobic membranes for use in human whole blood.

  19. Deposition of Lanthanum Strontium Cobalt Ferrite (LSCF) Using Suspension Plasma Spraying for Oxygen Transport Membrane Applications

    Science.gov (United States)

    Fan, E. S. C.; Kesler, O.

    2015-08-01

    Suspension plasma spray deposition was utilized to fabricate dense lanthanum strontium cobalt ferrite oxygen separation membranes (OSMs) on porous metal substrates for mechanical support. The as-sprayed membranes had negligible and/or reversible material decomposition. At the longer stand-off distance (80 mm), smooth and dense membranes could be manufactured using a plasma with power below approximately 81 kW. Moreover, a membrane of 55 μm was observed to have very low gas leakage rates desirable for OSM applications. This thickness could potentially be decreased further to improve oxygen diffusion by using metal substrates with finer surface pores.

  20. Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.

    Science.gov (United States)

    Yeats, Trevor H; Bacic, Antony; Johnson, Kim L

    2018-04-18

    Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.

  1. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  2. [Interaction of FABP4 with plasma membrane proteins of endothelial cells].

    Science.gov (United States)

    Saavedra, Paula; Girona, Josefa; Aragonès, Gemma; Cabré, Anna; Guaita, Sandra; Heras, Mercedes; Masana, Lluís

    2015-01-01

    Fatty acid binding protein (FABP4) is an adipose tissue-secreted adipokine implicated in the regulation of the energetic metabolism and inflammation. High levels of circulating FABP4 have been described in people with obesity, atherogenic dyslipidemia, diabetes and metabolic syndrome. Recent studies have demonstrated that FABP4 could have a direct effect on peripheral tissues and, specifically, on vascular function. It is still unknown how the interaction between FABP4 and the endothelial cells is produced to prompt these effects on vascular function. The objective of this work is studying the interaction between FABP4 and the plasma membrane proteins of endothelial cells. HUVEC cells were incubated with and without FABP4 (100 ng/ml) for 5 minutes. Immunolocalization of FABP4 was studied by confocal microscopy. The results showed that FABP4 colocalizates with CD31, a membrane protein marker. A strategy which combines 6XHistidine-tag FABP4 (FABP4-His), incubations with or without FABP4-His (100 ng/ml), formaldehyde cross-linking, cellular membrane protein extraction and western blot, was designed to study the FABP4 interactions with membrane proteins of HUVECs. The results showed different western blot profiles depending of the incubation with or without FABP4-His. The immunoblot revelead three covalent protein complexes of about 108, 77 and 33 kDa containing FAPB4 and its putative receptor. The existence of a specific binding protein complex able to bind FABP4 to endothelial cells is supported by these results. The obtained results will permit us advance in the molecular knowledge of FABP4 effects as well as use this protein and its receptor as therapeutic target to prevent cardiovascular. Copyright © 2014 Sociedad Española de Arteriosclerosis. Published by Elsevier España. All rights reserved.

  3. Comparative proteomic analysis of plasma membrane proteins between human osteosarcoma and normal osteoblastic cell lines

    International Nuclear Information System (INIS)

    Zhang, Zhiyu; Ma, Fang; Cai, Zhengdong; Zhang, Lijun; Hua, Yingqi; Jia, Xiaofang; Li, Jian; Hu, Shuo; Peng, Xia; Yang, Pengyuan; Sun, Mengxiong

    2010-01-01

    Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. However, the knowledge in diagnostic modalities has progressed less. To identify new biomarkers for the early diagnosis of OS as well as for potential novel therapeutic candidates, we performed a sub-cellular comparative proteomic research. An osteosarcoma cell line (MG-63) and human osteoblastic cells (hFOB1.19) were used as our comparative model. Plasma membrane (PM) was obtained by aqueous two-phase partition. Proteins were analyzed through iTRAQ-based quantitative differential LC/MS/MS. The location and function of differential proteins were analyzed through GO database. Protein-protein interaction was examined through String software. One of differentially expressed proteins was verified by immunohistochemistry. 342 non-redundant proteins were identified, 68 of which were differentially expressed with 1.5-fold difference, with 25 up-regulated and 43 down-regulated. Among those differential proteins, 69% ware plasma membrane, which are related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and interaction with each other. One protein--CD151 located in net nodes was verified to be over-expressed in osteosarcoma tissue by immunohistochemistry. It is the first time to use plasma membrane proteomics for studying the OS membrane proteins according to our knowledge. We generated preliminary but comprehensive data about membrane protein of osteosarcoma. Among these, CD151 was further validated in patient samples, and this small molecule membrane might be a new target for OS research. The plasma membrane proteins identified in this study may provide new insight into osteosarcoma biology and potential diagnostic and therapeutic biomarkers

  4. A method to modify PVDF microfiltration membrane via ATRP with low-temperature plasma pretreatment

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yu [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Ningbo University of Technology, Fenghua Road 201, Ningbo, 315211 (China); Song, Shuijun [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Zhejiang University of Science Technology, Liuhe Road 318, Hangzhou, 310023 (China); Lu, Yin, E-mail: luyin@nbu.edu.cn [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China); Zhu, Dongfa [School of Marine Science, Ningbo University, Fenghua Road 818, Ningbo, 315211 (China)

    2016-08-30

    Highlights: • We report a simple method to modify hydrophobic PVDF modification membrane. • Surface modification of PVDF membrane via ATRP with plasma pre-treatment. • ATRP grafting of SBMA onto the PVDF membrane surface form PVDF-g-SBMA membrane. • PVDF-g-SBMA membrane shows superior antifouling properties and hydrophilic. - Abstract: The hydrophilic modification of a polyvinylidene fluoride (PVDF) microfiltration membrane via pretreatment with argon plasma and direct surface-initiated atom transfer radical polymerization (ATRP) was studied. Both modified and unmodified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and pore size distribution measurements. FTIR and XPS spectra confirmed that sulfobetaine methacrylate (SBMA) had been grafted onto the membrane surface. The initial contact angle decreased from 87.0° to 29.8° and a water drop penetrated into the modified membrane completely in 8 s. The pore size distribution of the modified membrane exhibited a smaller mean value than that of the original membrane. The antifouling properties of the modified PVDF membrane were evaluated by a filtration test using bovine serum albumin (BSA) solution. The results showed that the initial flux of the modified membrane increased from 2140.1 L/m{sup 2} h to 2812.7 L/m{sup 2} h and the equilibrium flux of BSA solution increased from 31 L/m{sup 2} h to 53 L/m{sup 2} h.

  5. A method to modify PVDF microfiltration membrane via ATRP with low-temperature plasma pretreatment

    International Nuclear Information System (INIS)

    Han, Yu; Song, Shuijun; Lu, Yin; Zhu, Dongfa

    2016-01-01

    Highlights: • We report a simple method to modify hydrophobic PVDF modification membrane. • Surface modification of PVDF membrane via ATRP with plasma pre-treatment. • ATRP grafting of SBMA onto the PVDF membrane surface form PVDF-g-SBMA membrane. • PVDF-g-SBMA membrane shows superior antifouling properties and hydrophilic. - Abstract: The hydrophilic modification of a polyvinylidene fluoride (PVDF) microfiltration membrane via pretreatment with argon plasma and direct surface-initiated atom transfer radical polymerization (ATRP) was studied. Both modified and unmodified PVDF membranes were characterized by Fourier transform infrared spectroscopy (FTIR), water contact angle, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and pore size distribution measurements. FTIR and XPS spectra confirmed that sulfobetaine methacrylate (SBMA) had been grafted onto the membrane surface. The initial contact angle decreased from 87.0° to 29.8° and a water drop penetrated into the modified membrane completely in 8 s. The pore size distribution of the modified membrane exhibited a smaller mean value than that of the original membrane. The antifouling properties of the modified PVDF membrane were evaluated by a filtration test using bovine serum albumin (BSA) solution. The results showed that the initial flux of the modified membrane increased from 2140.1 L/m"2 h to 2812.7 L/m"2 h and the equilibrium flux of BSA solution increased from 31 L/m"2 h to 53 L/m"2 h.

  6. Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

    Science.gov (United States)

    Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

    2000-01-01

    BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

  7. Specific interaction of postsynaptic densities with membrane rafts isolated from synaptic plasma membranes.

    Science.gov (United States)

    Liu, Qian; Yao, Wei-Dong; Suzuki, Tatsuo

    2013-06-01

    Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. We recently demonstrated the presence, at the electron microscopic level, of complexes consisting of membrane rafts and postsynaptic densities (PSDs) in detergent-resistant membranes (DRMs) prepared from synaptic plasma membranes (SPMs) ( Suzuki et al., 2011 , J Neurochem, 119, 64-77). To further explore these complexes, here we investigated the nature of the binding between purified SPM-DRMs and PSDs in vitro. In binding experiments, we used SPM-DRMs prepared after treating SPMs with n-octyl-β-d-glucoside, because at concentrations of 1.0% or higher it completely separates SPM-DRMs and PSDs, providing substantially PSD-free unique SPM-DRMs as well as DRM-free PSDs. PSD binding to PSD-free DRMs was identified by mass spectrometry, Western blotting, and electron microscopy. PSD proteins were not incorporated into SPMs, and significantly less PSD proteins were incorporated into DRMs prepared from liver membranes, providing in vitro evidence that binding of PSDs to DRMs is specific and suggestion of the presence of specific interacting molecules. These specific interactions may have important roles in synaptic development, function, and plasticity in vivo. In addition, the binding system we developed may be a good tool to search for binding molecules and binding mechanisms between PSDs and rafts.

  8. Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation.

    Science.gov (United States)

    Chang, Yung; Shih, Yu-Ju; Ko, Chao-Yin; Jhong, Jheng-Fong; Liu, Ying-Ling; Wei, Ta-Chin

    2011-05-03

    In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.

  9. Assay of Plasma Membrane H+-ATPase in Plant Tissues under Abiotic Stresses.

    Science.gov (United States)

    Janicka, Małgorzata; Wdowikowska, Anna; Kłobus, Grażyna

    2018-01-01

    Plasma membrane (PM) H + -ATPase, which generates the proton gradient across the outer membrane of plant cells, plays a fundamental role in the regulation of many physiological processes fundamental for growth and development of plants. It is involved in the uptake of nutrients from external solutions, their loading into phloem and long-distance transport, stomata aperture and gas exchange, pH homeostasis in cytosol, cell wall loosening, and cell expansion. The crucial role of the enzyme in resistance of plants to abiotic and biotic stress factors has also been well documented. Such great diversity of physiological functions linked to the activity of one enzyme requires a suitable and complex regulation of H + -ATPase. This regulation comprises the transcriptional as well as post-transcriptional levels. Herein, we describe the techniques that can be useful for the analysis of the plasma membrane proton pump modifications at genetic and protein levels under environmental factors.

  10. NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes*

    Science.gov (United States)

    Cirillo, Federica; Ghiroldi, Andrea; Fania, Chiara; Piccoli, Marco; Torretta, Enrica; Tettamanti, Guido; Gelfi, Cecilia; Anastasia, Luigi

    2016-01-01

    NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking. PMID:26987901

  11. Lateral mobility of plasma membrane lipids in Xenopus eggs: regional differences related to animal/vegetal polarity become extreme upon fertilization.

    Science.gov (United States)

    Dictus, W J; van Zoelen, E J; Tetteroo, P A; Tertoolen, L G; de Laat, S W; Bluemink, J G

    1984-01-01

    Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-(N-hexadecanoyl)-aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (approximately 50%) has a fivefold smaller lateral diffusion coefficient (D = 1.5 X 10(-8) cm2/sec) in the animal than in the vegetal plasma membrane (D = 7.6 X 10(-8) cm2/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (greater than 100X) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements (D less than 10(-10) cm2/sec), whereas at the vegetal side D is only slightly reduced (D = 4.4 X 10(-8) cm2/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.

  12. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

    NARCIS (Netherlands)

    Laat, S.W. de; Tetteroo, P.A.T.; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van

    1984-01-01

    The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xaopus Levis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-(N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-(N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The

  14. The Plasma Membrane of Saccharomyces cerevisiae : Structure, Function, and Biogenesis

    NARCIS (Netherlands)

    VANDERREST, ME; KAMMINGA, AH; NAKANO, A; ANRAKU, Y; POOLMAN, B; KONINGS, WN

    The composition of phospholipids, sphingolipids, and sterols in the plasma membrane has a strong influence on the activity of the proteins associated or embedded in the lipid bilayer. Since most lipid-synthesizing enzymes in Saccharomyces cerevisiae are located in intracellular organelles, an

  15. Metabolism of phosphatidylinositol in plasma membranes and synaptosomes of rat cerebral cortex: A comparison between endogenous vs exogenous substrate pools

    International Nuclear Information System (INIS)

    Navidi, M.; MacQuarrie, R.A.; Sun, G.Y.

    1990-01-01

    The metabolism of phosphatidylinositols (PI) labeled with [14C]arachidonic acid within plasma membranes or synaptosomes was compared to the metabolism of PI prelabeled with [14C]arachidonic acid and added exogenously to the same membranes. Incubation of membranes containing the endogenously-labeled PI pool in the presence of Ca2+ resulted in the release of labeled arachidonic acid, as well as a small amount of labeled diacylglycerol. Labeled arachidonic acid was effectively reutilized and returned to the membrane phospholipids in the presence of adenosine triphosphate (ATP), CoA, and lysoPI. Although Ca2+ promoted the release of labeled diacylglycerol from prelabeled plasma membranes, this amount was only 17% of the maximal release, i.e., release in the presence of deoxycholate and Ca2+. This latter condition is known to fully activate the PI-phospholipase C, and incubation of prelabeled plasma membranes resulted in a six-fold increase in labeled diacylglycerols. On the other hand, when exogenously labeled PI were incubated with plasma membranes in the presence of Ca2+, the labeled diacylglycerols released were 59% of that compared to the fully activated condition. The phospholipase C action was calcium-dependent, regardless of whether exogenous or endogenous substrates were used in the incubation. In contrast to plasma membranes, intact synaptosomes had limited ability to metabolize exogenous PI even in the presence of Ca2+, although the activity of phospholipase C was similar to that in the plasma membranes when assayed in the presence of deoxycholate and Ca2+. These results suggest that discrete pools of PI are present in plasma membranes, and that the pool associated with the acyltransferase is apparently not readily accessible to hydrolysis by phospholipase C

  16. Confining Domains Lead to Reaction Bursts: Reaction Kinetics in the Plasma Membrane

    Science.gov (United States)

    Kalay, Ziya; Fujiwara, Takahiro K.; Kusumi, Akihiro

    2012-01-01

    Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity. PMID:22479350

  17. Confining domains lead to reaction bursts: reaction kinetics in the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Ziya Kalay

    Full Text Available Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity.

  18. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork.

    Science.gov (United States)

    Lenne, Pierre-François; Wawrezinieck, Laure; Conchonaud, Fabien; Wurtz, Olivier; Boned, Annie; Guo, Xiao-Jun; Rigneault, Hervé; He, Hai-Tao; Marguet, Didier

    2006-07-26

    It is by now widely recognized that cell membranes show complex patterns of lateral organization. Two mechanisms involving either a lipid-dependent (microdomain model) or cytoskeleton-based (meshwork model) process are thought to be responsible for these plasma membrane organizations. In the present study, fluorescence correlation spectroscopy measurements on various spatial scales were performed in order to directly identify and characterize these two processes in live cells with a high temporal resolution, without any loss of spatial information. Putative raft markers were found to be dynamically compartmented within tens of milliseconds into small microdomains (Ø confinement of the transferrin receptor protein. A free-like diffusion was observed when both the lipid-dependent and cytoskeleton-based organizations were disrupted, which suggests that these are two main compartmentalizing forces at work in the plasma membrane.

  19. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    International Nuclear Information System (INIS)

    Ebner, Andreas; Hinterdorfer, Peter; Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann; Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika

    2008-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl - ) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients

  20. Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

    Energy Technology Data Exchange (ETDEWEB)

    Ebner, Andreas; Hinterdorfer, Peter [Institute for Biophysics, University of Linz, A-4040 Linz (Austria); Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann [Institute of Physiology II, University of Muenster, D-48149 Muenster (Germany); Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika [Department of Pediatrics, University Hospitals of Muenster, D-48149 Muenster (Germany)], E-mail: schille@uni-muenster.de

    2008-09-24

    CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl{sup -}) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

  1. The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

    Science.gov (United States)

    Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

    2011-01-01

    The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

  2. GLUT4 expression at the plasma membrane is related to fibre volume in human skeletal muscle fibres

    DEFF Research Database (Denmark)

    Gaster, M; Vach, W; Beck-Nielsen, H

    2002-01-01

    In this study we examined the relationship between GLUT4 expression at the plasma membrane and muscle fibre size in fibre-typed human muscle fibres by immunocytochemistry and morphometry in order to gain further insight into the regulation of GLUT4 expression. At the site of the plasma membrane...

  3. Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

    Science.gov (United States)

    Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

    2013-07-01

    Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

  4. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

    Science.gov (United States)

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

    2015-06-01

    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  5. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Magini, Alessandro [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Polchi, Alice; Urbanelli, Lorena [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Cesselli, Daniela; Beltrami, Antonio [Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Tancini, Brunella [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Emiliani, Carla, E-mail: carla.emiliani@unipg.it [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  6. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    International Nuclear Information System (INIS)

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-01-01

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane

  7. Uniform Structure of Eukaryotic Plasma Membrane: Lateral Domains in Plants

    Czech Academy of Sciences Publication Activity Database

    Malínská, Kateřina; Zažímalová, Eva

    2011-01-01

    Roč. 12, č. 2 (2011), s. 148-155 ISSN 1389-2037 R&D Projects: GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : Plasma membrane * microdomains * lateral segregation Subject RIV: ED - Physiology Impact factor: 2.886, year: 2011

  8. Effect of washing on the plasma membrane and on stress reactions of cultured rose cells

    International Nuclear Information System (INIS)

    Qian, Y.C.; Nguyen, T.; Murphy, T.M.

    1993-01-01

    Cultured cells of Rosa damascena have been used as a model for studies of responses of plant cells to various stresses, including UV radiation, protein-synthesis inhibitors, and elicitors from pathogens. Many of the responses involve reactions at the plasma membrane: efflux of K + , changes in the acid balance between cytoplasm and external medium, synthesis of H 2 O 2 , and inhibition of ferricyanide reduction. In previous studies, the cells have typically been washed with a solution of low ionic strength. We now show that this washing procedure results in changes in the protein composition of the plasma membrane, in the labeling of the proteins in the plasma membrane, and in the specific activity of ATPase in purified plasma membrane vesicles. Also, compared to the unwashed cells, the washed cells show less net K + efflux after UV-C and Phytophthora elicitor treatments; more synthesis of H 2 O 2 after UV-C and a pattern of accumulation of H 2 O 2 after elicitor treatment that shows a delayed but higher peak; and more inhibition of ferricyanide reduction after UV-C, but not after elicitor treatment. The results suggest that washing has differential effects on the mechanisms by which cultured plant cells perceive or respond to two stresses, UV-C and elicitor

  9. Amine Enrichment of Thin-Film Composite Membranes via Low Pressure Plasma Polymerization for Antimicrobial Adhesion.

    Science.gov (United States)

    Reis, Rackel; Dumée, Ludovic F; He, Li; She, Fenghua; Orbell, John D; Winther-Jensen, Bjorn; Duke, Mikel C

    2015-07-15

    Thin-film composite membranes, primarily based on poly(amide) (PA) semipermeable materials, are nowadays the dominant technology used in pressure driven water desalination systems. Despite offering superior water permeation and salt selectivity, their surface properties, such as their charge and roughness, cannot be extensively tuned due to the intrinsic fabrication process of the membranes by interfacial polymerization. The alteration of these properties would lead to a better control of the materials surface zeta potential, which is critical to finely tune selectivity and enhance the membrane materials stability when exposed to complex industrial waste streams. Low pressure plasma was employed to introduce amine functionalities onto the PA surface of commercially available thin-film composite (TFC) membranes. Morphological changes after plasma polymerization were analyzed by SEM and AFM, and average surface roughness decreased by 29%. Amine enrichment provided isoelectric point changes from pH 3.7 to 5.2 for 5 to 15 min of plasma polymerization time. Synchrotron FTIR mappings of the amine-modified surface indicated the addition of a discrete 60 nm film to the PA layer. Furthermore, metal affinity was confirmed by the enhanced binding of silver to the modified surface, supported by an increased antimicrobial functionality with demonstrable elimination of E. coli growth. Essential salt rejection was shown minimally compromised for faster polymerization processes. Plasma polymerization is therefore a viable route to producing functional amine enriched thin-film composite PA membrane surfaces.

  10. Controlled change of transport properties of poly(ethylene terephthalate) track membranes by plasma method

    International Nuclear Information System (INIS)

    Kravets, L I; Dmitriev, S N; Drachev, A I; Gilman, A B; Lazea, A; Dinescu, G

    2007-01-01

    A process of plasma polymerization of dimethylaniline and acrylic acid vapours on the surface of poly(ethylene terephthalate) track membranes has been investigated. The surface and hydrodynamic properties of the composite membranes produced in this case have been studied. It is shown that the water permeability of the obtained polymeric membranes can be controlled by changing the filtrate pH. Membranes with such properties can be used for controllable drug delivery and in sensor control

  11. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts. Summary progress report, May 16, 1987--June 1, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1991-12-31

    This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

  12. ER-plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis.

    Science.gov (United States)

    Nascimbeni, Anna Chiara; Giordano, Francesca; Dupont, Nicolas; Grasso, Daniel; Vaccaro, Maria I; Codogno, Patrice; Morel, Etienne

    2017-07-14

    The double-membrane-bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl-inositol-3-phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER-plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E-Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E-Syt-containing domains during autophagy and that inhibition of E-Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E-Syts are essential for autophagy-associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER-plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy. © 2017 The Authors.

  13. ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology.

    Science.gov (United States)

    Manford, Andrew G; Stefan, Christopher J; Yuan, Helen L; Macgurn, Jason A; Emr, Scott D

    2012-12-11

    Endoplasmic reticulum-plasma membrane (ER-PM) junctions are conserved structures defined as regions of the ER that tightly associate with the plasma membrane. However, little is known about the mechanisms that tether these organelles together and why such connections are maintained. Using a quantitative proteomic approach, we identified three families of ER-PM tethering proteins in yeast: Ist2 (related to mammalian TMEM16 ion channels), the tricalbins (Tcb1/2/3, orthologs of the extended synaptotagmins), and Scs2 and Scs22 (vesicle-associated membrane protein-associated proteins). Loss of all six tethering proteins results in the separation of the ER from the PM and the accumulation of cytoplasmic ER. Importantly, we find that phosphoinositide signaling is misregulated at the PM, and the unfolded protein response is constitutively activated in the ER in cells lacking ER-PM tether proteins. These results reveal critical roles for ER-PM contacts in cell signaling, organelle morphology, and ER function. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

    Science.gov (United States)

    Chevalier, Adrien S; Chaumont, François

    2015-05-01

    Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Endogenous glycosphingolipid acceptor specificity of sialosyltransferase systems in intact golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain

    International Nuclear Information System (INIS)

    Durrie, R.; Saito, M.; Rosenberg, A.

    1988-01-01

    Preparations highly enriched in Golgi complex membranes, synaptosomes, and synaptic plasma membranes (SPM) by marker enzyme analysis and electron microscopic morphology were made from the brains of 28-day-old rats. These were incubated with cytidine 5'-monophosphate-N-acetyl[ 14 C]neuraminic acid (CMP-NeuAc) in a physiologic buffer, without detergents. Glycolipid sialosyltransferase activities (SATs) were measured by analyzing incorporation of radiolabeled NeuAc into endogenous membrane gangliosides. Golgi SAT was diversified in producing all the various molecular species of labeled gangliosides. Synaptosomal SAT exhibited a lower activity, but it was highly specific in its labeling pattern, with a marked preference for labeling NeuAcα2 → 8NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer (GD3 ganglioside). SPM prepared from the synaptosomes retained the GD3-related SAT (or SAT-2), and the total specific activity increased, which suggests that the location of the synaptosomal activity is in the SPM. These results indicate that SAT activity in Golgi membranes differs from that in synaptosomes with regard to endogenous acceptor substrate specificity and SAT activity of synaptosomes should be located in the synaptosomal plasma membrane. This SAT could function as an ectoenzyme in concert with ecto-sialidase to modulate the GD3 and other ganglioside population in situ at the SPM of the central nervous system

  16. Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration.

    Science.gov (United States)

    Volkov, A V; Tsarkov, S E; Gilman, A B; Khotimsky, V S; Roldughin, V I; Volkov, V V

    2015-08-01

    For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Tissue-specific expression of the gene for a putative plasma membrane H(+)-ATPase in a seagrass.

    Science.gov (United States)

    Fukuhara, T; Pak, J Y; Ohwaki, Y; Tsujimura, H; Nitta, T

    1996-01-01

    A cDNA clone corresponding to the gene (ZHA1) for a putative plasma membrane H(+)-ATPase of a seagrass (Zostera marina L.) was isolated and sequenced. Comparison of the amino acid predicted sequence from the nucleotide sequence of ZHA1 with those encoded by known genes for plasma membrane H(+)-ATPases from other plants indicated that ZHA1 is most similar to the gene (PMA4) for a plasma membrane H(+)-ATPase in a tobacco (84.4%). Northern hybridization indicated that ZHA1 was strongly expressed in mature leaves, which are exposed to seawater and have the ability of tolerate salinity; ZHA1 was weakly expressed in immature leaves, which are protected from seawater by tightly enveloping sheaths and are sensitive to salinity. In mature leaves, in situ hybridization revealed that ZHA1 was expressed specifically in epidermal cells, the plasma membranes of which were highly invaginated and morphologically similar to those of typical transfer cells. Therefore, the differentiation of the transfer cell-like structures, accompanied by the high-level expression of ZHA1, in the epidermal cells of mature leaves in particular may be important for the excretion of salt by these cells. PMID:8587992

  18. In Vitro Dialysis of Cytokine-Rich Plasma With High and Medium Cut-Off Membranes Reduces Its Procalcific Activity.

    Science.gov (United States)

    Willy, Kevin; Hulko, Michael; Storr, Markus; Speidel, Rose; Gauss, Julia; Schindler, Ralf; Zickler, Daniel

    2017-09-01

    Recently developed high-flux (HF) dialysis membranes with extended permeability provide better clearance of middle-sized molecules such as interleukins (ILs). Whether this modulation of inflammation influences the procalcific effects of septic plasma on vascular smooth muscle cells (VSMCs) is not known. To assess the effects of high cut-off (HCO) and medium cut-off (MCO) membranes on microinflammation and in vitro vascular calcification we developed a miniature dialysis model. Plasma samples from lipopolysaccharide-spiked blood were dialyzed with HF, HCO, and MCO membranes in an in vitro miniature dialysis model. Afterwards, IL-6 concentrations were determined in dialysate and plasma. Calcifying VSMCs were incubated with dialyzed plasma samples and vascular calcification was assessed. Osteopontin (OPN) and matrix Gla protein (MGP) were measured in VSMC supernatants. IL-6 plasma concentrations were markedly lower with HCO and MCO dialysis. VSMC calcification was significantly lower after incubation with MCO- and HCO-serum compared to HF plasma. MGP and OPN levels in supernatants were significantly lower in the MCO but not in the HCO group compared to HF. In vitro dialysis of cytokine-enriched plasma samples with MCO and HCO membranes reduces IL-6 levels. The induction of vascular calcification by cytokine-enriched plasma is reduced after HCO and MCO dialysis. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

  19. Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

    Science.gov (United States)

    Kiechle, F L; Sykes, E; Artiss, J D

    1995-01-01

    Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

  20. Confinement of activating receptors at the plasma membrane controls natural killer cell tolerance.

    Science.gov (United States)

    Guia, Sophie; Jaeger, Baptiste N; Piatek, Stefan; Mailfert, Sébastien; Trombik, Tomasz; Fenis, Aurore; Chevrier, Nicolas; Walzer, Thierry; Kerdiles, Yann M; Marguet, Didier; Vivier, Eric; Ugolini, Sophie

    2011-04-05

    Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

  1. Fluorescence Recovery After Photobleaching Analysis of the Diffusional Mobility of Plasma Membrane Proteins: HER3 Mobility in Breast Cancer Cell Membranes.

    Science.gov (United States)

    Sarkar, Mitul; Koland, John G

    2016-01-01

    The fluorescence recovery after photobleaching (FRAP) method is a straightforward means of assessing the diffusional mobility of membrane-associated proteins that is readily performed with current confocal microscopy instrumentation. We describe here the specific application of the FRAP method in characterizing the lateral diffusion of genetically encoded green fluorescence protein (GFP)-tagged plasma membrane receptor proteins. The method is exemplified in an examination of whether the previously observed segregation of the mammalian HER3 receptor protein in discrete plasma membrane microdomains results from its physical interaction with cellular entities that restrict its mobility. Our FRAP measurements of the diffusional mobility of GFP-tagged HER3 reporters expressed in MCF7 cultured breast cancer cells showed that despite the observed segregation of HER3 receptors within plasma membrane microdomains their diffusion on the macroscopic scale is not spatially restricted. Thus, in FRAP analyses of various HER3 reporters a near-complete recovery of fluorescence after photobleaching was observed, indicating that HER3 receptors are not immobilized by long-lived physical interactions with intracellular species. An examination of HER3 proteins with varying intracellular domain sequence truncations also indicated that a proposed formation of oligomeric HER3 networks, mediated by physical interactions involving specific HER3 intracellular domain sequences, either does not occur or does not significantly reduce HER3 mobility on the macroscopic scale.

  2. Silver ions increase plasma membrane permeability through modulation of intracellular calcium levels in tobacco BY-2 cells.

    Science.gov (United States)

    Klíma, Petr; Laňková, Martina; Vandenbussche, Filip; Van Der Straeten, Dominique; Petrášek, Jan

    2018-05-01

    Silver ions increase plasma membrane permeability for water and small organic compounds through their stimulatory effect on plasma membrane calcium channels, with subsequent modulation of intracellular calcium levels and ion homeostasis. The action of silver ions at the plant plasma membrane is largely connected with the inhibition of ethylene signalling thanks to the ability of silver ion to replace the copper cofactor in the ethylene receptor. A link coupling the action of silver ions and cellular auxin efflux has been suggested earlier by their possible direct interaction with auxin efflux carriers or by influencing plasma membrane permeability. Using tobacco BY-2 cells, we demonstrate here that besides a dramatic increase of efflux of synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthalene acetic acid (NAA), treatment with AgNO 3 resulted in enhanced efflux of the cytokinin trans-zeatin (tZ) as well as the auxin structural analogues tryptophan (Trp) and benzoic acid (BA). The application of AgNO 3 was accompanied by gradual water loss and plasmolysis. The observed effects were dependent on the availability of extracellular calcium ions (Ca 2+ ) as shown by comparison of transport assays in Ca 2+ -rich and Ca 2+ -free buffers and upon treatment with inhibitors of plasma membrane Ca 2+ -permeable channels Al 3+ and ruthenium red, both abolishing the effect of AgNO 3 . Confocal microscopy of Ca 2+ -sensitive fluorescence indicator Fluo-4FF, acetoxymethyl (AM) ester suggested that the extracellular Ca 2+ availability is necessary to trigger the response to silver ions and that the intracellular Ca 2+ pool alone is not sufficient for this effect. Altogether, our data suggest that in plant cells the effects of silver ions originate from the primal modification of the internal calcium levels, possibly by their interaction with Ca 2+ -permeable channels at the plasma membrane.

  3. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    International Nuclear Information System (INIS)

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.

    1987-01-01

    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size

  4. Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

    Science.gov (United States)

    Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R

    2018-01-01

    A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

  5. Advanced spinning disk-TIRF microscopy for faster imaging of the cell interior and the plasma membrane.

    Science.gov (United States)

    Zobiak, Bernd; Failla, Antonio Virgilio

    2018-03-01

    Understanding the cellular processes that occur between the cytosol and the plasma membrane is an important task for biological research. Till now, however, it was not possible to combine fast and high-resolution imaging of both the isolated plasma membrane and the surrounding intracellular volume. Here, we demonstrate the combination of fast high-resolution spinning disk (SD) and total internal reflection fluorescence (TIRF) microscopy for specific imaging of the plasma membrane. A customised SD-TIRF microscope was used with specific design of the light paths that allowed, for the first time, live SD-TIRF experiments at high acquisition rates. A series of experiments is shown to demonstrate the feasibility and performance of our setup. © 2017 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

  6. Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

    Science.gov (United States)

    Jones, Roy; James, Peter S; Howes, Liz; Bruckbauer, Andreas; Klenerman, David

    2007-07-01

    In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

  7. Plant Defense Response to Fungal Pathogens (Activation of Host-Plasma Membrane H+-ATPase by Elicitor-Induced Enzyme Dephosphorylation).

    Science.gov (United States)

    Vera-Estrella, R.; Barkla, B. J.; Higgins, V. J.; Blumwald, E.

    1994-01-01

    Elicitor preparations containing the avr5 gene products from race 4 of Cladosporium fulvum and tomato (Lycopersicon esculentum L.) cells near isogenic for the resistance gene Cf5 were used to investigate events following the treatment of host plasma membranes with elicitor. A 4-fold increase in H+-ATPase activity, coincident with the acidification of the extracellular medium, was detected immediately after elicitor treatment. The elicitor-induced stimulation of the plasma membrane H+-ATPase was inhibited by okadaic acid but not by staurosporine, suggesting that protein dephosphorylation was required for increased H+-ATPase activity. This observation was confirmed by [gamma]-32P labeling and immunodetection of the plasma membrane H+-ATPase. Effects of guanidine nucleotide analogs and mastoparan on the ATPase activity suggested the role of GTP-binding proteins in mediating the putative elicitor-receptor binding, resulting in activation of a phosphatase(s), which in turn stimulates the plasma membrane H+-ATPase by dephosphorylation. PMID:12232073

  8. Positive zeta potential of a negatively charged semi-permeable plasma membrane

    Science.gov (United States)

    Sinha, Shayandev; Jing, Haoyuan; Das, Siddhartha

    2017-08-01

    The negative charge of the plasma membrane (PM) severely affects the nature of moieties that may enter or leave the cells and controls a large number of ion-interaction-mediated intracellular and extracellular events. In this letter, we report our discovery of a most fascinating scenario, where one interface (e.g., membrane-cytosol interface) of the negatively charged PM shows a positive surface (or ζ) potential, while the other interface (e.g., membrane-electrolyte interface) still shows a negative ζ potential. Therefore, we encounter a completely unexpected situation where an interface (e.g., membrane-cytosol interface) that has a negative surface charge density demonstrates a positive ζ potential. We establish that the attainment of such a property by the membrane can be ascribed to an interplay of the nature of the membrane semi-permeability and the electrostatics of the electric double layer established on either side of the charged membrane. We anticipate that such a membrane property can lead to such capabilities of the cell (in terms of accepting or releasing certain kinds of moieties as well regulating cellular signaling) that was hitherto inconceivable.

  9. Super-resolution microscopy reveals the insulin-resistance-regulated reorganization of GLUT4 on plasma membranes.

    Science.gov (United States)

    Gao, Lan; Chen, Junling; Gao, Jing; Wang, Hongda; Xiong, Wenyong

    2017-01-15

    GLUT4 (also known as SLC2A4) is essential for glucose uptake in skeletal muscles and adipocytes, which play central roles in whole-body glucose metabolism. Here, using direct stochastic optical reconstruction microscopy (dSTORM) to investigate the characteristics of plasma-membrane-fused GLUT4 at the single-molecule level, we have demonstrated that insulin and insulin resistance regulate the spatial organization of GLUT4 in adipocytes. Stimulation with insulin shifted the balance of GLUT4 on the plasma membrane toward a more dispersed configuration. In contrast, insulin resistance induced a more clustered distribution of GLUT4 and increased the mean number of molecules per cluster. Furthermore, our data demonstrate that the F 5 QQI motif and lipid rafts mediate the maintenance of GLUT4 clusters on the plasma membrane. Mutation of F 5 QQI (F 5 QQA-GLUT4) induced a more clustered distribution of GLUT4; moreover, destruction of lipid rafts in adipocytes expressing F 5 QQA-GLUT4 dramatically decreased the percentage of large clusters and the mean number of molecules per cluster. In conclusion, our data clarify the effects of insulin stimulation or insulin resistance on GLUT4 reorganization on the plasma membrane and reveal new pathogenic mechanisms of insulin resistance. © 2017. Published by The Company of Biologists Ltd.

  10. Surface Modification of Polypropylene Microporous Membrane by Atmospheric-Pressure Plasma Immobilization of N,N-dimethylamino Ethyl Methacrylate

    International Nuclear Information System (INIS)

    Zhong Shaofeng

    2010-01-01

    Surface modification of polypropylene microporous membrane (PPMM) was performed by atmospheric pressure dielectric barrier discharge plasma immobilization of N,N-dimethylamino ethyl methacrylate (DMAEMA). Structural and morphological changes on the membrane surface were characterized by attenuated total reflection-Fourier transform infrared spectroscopy (FT-IR/ATR), X-ray photoelectron spectroscope (XPS) and field emission scanning electron microscopy (FE-SEM). Water contact angles of the membrane surfaces were also measured by the sessile drop method. Results reveal that both the plasma-treating conditions and the adsorbed DMAEMA amount have remarkable effects on the immobilization degree of DMAEMA. Peroxide determination by 1,1-diphenyl-2-picrvlhydrazyl (DPPH) method verifies the exsistence of radicals induced by plasma, which activize the immobilization reaction. Pure water contact angle on the membrane surface decreased with the increase of DMAEMA immobilization degree, which indicates an enhanced hydrophilicity for the modified membranes. The effects of immobilization degrees on pure water fluxes were also measured. It is shown that pure water fluxes first increased with immobilization degree and then decreased. Finally, permeation of bovine serum albumin (BSA) and lysozyme solution were measured to evaluate the antifouling property of the DMAEMA-modified membranes, from which it is shown that both hydrophilicity and electrostatic repulsion are beneficial for membrane antifouling.

  11. Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

    Science.gov (United States)

    Minami, Anzu; Tominaga, Yoko; Furuto, Akari; Kondo, Mariko; Kawamura, Yukio; Uemura, Matsuo

    2015-08-01

    The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  12. Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity

    OpenAIRE

    Laat, S.W. de; Bluemink, J.G.; Dictus, W.J.A.G.; Zoelen, E.J.J. van; Tetteroo, P.A.T.; Tertoolen, L.G.J.

    1984-01-01

    Regional differences in the lateral mobility properties of plasma membrane lipids were studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein- -1abelled fatty acids HEDAF (5-(N-hexadecanoyl)- aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to distribute itself in the plasma membrane. Under all experimental conditions used these molecules s...

  13. Structural changes in plasma membranes prepared from irradiated Chinese hamster V79 cells as revealed by Raman spectroscopy

    International Nuclear Information System (INIS)

    Verma, S.P.; Sonwalkar, N.

    1991-01-01

    The effect of gamma irradiation on the integrity of plasma membranes isolated from Chinese hamster V79 cells was investigated by Raman spectroscopy. Plasma membranes of control V79 cells show transitions between -10 and 5 degree C (low-temperature transition), 10 and 22 degree C (middle-temperature transition), and 32 and 40 degree C (high-temperature transition). Irradiation (5 Gy) alters these transitions markedly. First, the low-temperature transition shifts to higher temperature (onset and completion temperatures 4 and 14 degree C). Second, the middle-temperature transition shifts up to the range of about 20-32 degree C, but the width remains unchanged. Third, the higher temperature transition broadens markedly and shifts to the range of about 15-40 degree C. Protein secondary structure as determined by least-squares analysis of the amide I bands shows 36% total helix, 55% total beta-strand, and 9% turn plus undefined for control plasma membrane proteins. Plasma membrane proteins of irradiated V79 cells show an increase in total helix (40 and 45% at 5 and 10 Gy, respectively) and a decrease in the total beta-strand (48 and 44% at 5 and 10 Gy, respectively) structures. The qualitative analysis of the Raman features of plasma membranes and model compounds in the 1600 cm-1 region, assigned to tyrosine groups, revealed that irradiation alters the microenvironment of these groups. We conclude that the radiation dose used in the survival range of Chinese hamster V79 cells can cause damage to plasma membrane proteins without detectable lipid peroxidation, and that the altered proteins react differently with lipids, yielding a shift in the thermal transition properties

  14. A hypothesis for the minimal overall structure of the mammalian plasma membrane redox system.

    Science.gov (United States)

    de Grey, Aubrey D N J

    2003-05-01

    After a long period of frustration, many components of the mammalian plasma membrane redox system are now being identified at the molecular level. Some are apparently ubiquitous but are necessary only for a subset of electron donors or acceptors; some are present only in certain cell types; some appear to be associated with proton extrusion; some appear to be capable of superoxide production. The volume and variety of data now available have begun to allow the formulation of tentative models for the overall network of interactions of enzymes and substrates that together make up the plasma membrane redox system. Such a model is presented here. The structure discussed here is of the mammalian system, though parts of it may apply more or less accurately to fungal and plant cells too. Judging from the history of mitochondrial oxidative phosphorylation, it may be hoped that the development of models of the whole system - even if they undergo substantial revision thereafter - will markedly accelerate the pace of research in plasma membrane redox, by providing a coherent basis for the design of future experiments.

  15. Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

    International Nuclear Information System (INIS)

    Wallace, A.V.; Martin, B.R.; Houslay, M.D.

    1990-01-01

    Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa

  16. Solid polymer electrolyte composite membrane comprising plasma etched porous support

    Science.gov (United States)

    Liu, Han; LaConti, Anthony B.

    2010-10-05

    A solid polymer electrolyte composite membrane and method of manufacturing the same. According to one embodiment, the composite membrane comprises a rigid, non-electrically-conducting support, the support preferably being a sheet of polyimide having a thickness of about 7.5 to 15 microns. The support has a plurality of cylindrical pores extending perpendicularly between opposing top and bottom surfaces of the support. The pores, which preferably have a diameter of about 0.1 to 5 microns, are made by plasma etching and preferably are arranged in a defined pattern, for example, with fewer pores located in areas of high membrane stress and more pores located in areas of low membrane stress. The pores are filled with a first solid polymer electrolyte, such as a perfluorosulfonic acid (PFSA) polymer. A second solid polymer electrolyte, which may be the same as or different than the first solid polymer electrolyte, may be deposited over the top and/or bottom of the first solid polymer electrolyte.

  17. Prevention of intra-abdominal adhesion by bi-layer electrospun membrane.

    Science.gov (United States)

    Jiang, Shichao; Wang, Wei; Yan, Hede; Fan, Cunyi

    2013-06-04

    The aim of this study was to compare the anti-adhesion efficacy of a bi-layer electrospun fibrous membrane consisting of hyaluronic acid-loaded poly(ε-caprolactone) (PCL) fibrous membrane as the inner layer and PCL fibrous membrane as the outer layer with a single-layer PCL electrospun fibrous membrane in a rat cecum abrasion model. The rat model utilized a cecal abrasion and abdominal wall insult surgical protocol. The bi-layer and PCL membranes were applied between the cecum and the abdominal wall, respectively. Control animals did not receive any treatment. After postoperative day 14, a visual semiquantitative grading scale was used to grade the extent of adhesion. Histological analysis was performed to reveal the features of adhesion tissues. Bi-layer membrane treated animals showed significantly lower adhesion scores than control animals (p compared with the PCL membrane. Histological analysis of the bi-layer membrane treated rat rarely demonstrated tissue adhesion while that of the PCL membrane treated rat and control rat showed loose and dense adhesion tissues, respectively. Bi-layer membrane can efficiently prevent adhesion formation in abdominal cavity and showed a significantly decreased adhesion tissue formation compared with the control.

  18. Numerical calculation on a two-step subdiffusion behavior of lateral protein movement in plasma membranes

    Science.gov (United States)

    Sumi, Tomonari; Okumoto, Atsushi; Goto, Hitoshi; Sekino, Hideo

    2017-10-01

    A two-step subdiffusion behavior of lateral movement of transmembrane proteins in plasma membranes has been observed by using single-molecule experiments. A nested double-compartment model where large compartments are divided into several smaller ones has been proposed in order to explain this observation. These compartments are considered to be delimited by membrane-skeleton "fences" and membrane-protein "pickets" bound to the fences. We perform numerical simulations of a master equation using a simple two-dimensional lattice model to investigate the heterogeneous diffusion dynamics behavior of transmembrane proteins within plasma membranes. We show that the experimentally observed two-step subdiffusion process can be described using fence and picket models combined with decreased local diffusivity of transmembrane proteins in the vicinity of the pickets. This allows us to explain the two-step subdiffusion behavior without explicitly introducing nested double compartments.

  19. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

  20. A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

    2011-01-01

    transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase...... maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...

  1. Exploring in vivo cholesterol-mediated interactions between activated EGF receptors in plasma membrane with single-molecule optical tracking

    International Nuclear Information System (INIS)

    Lin, Chien Y.; Huang, Jung Y.; Lo, Leu-Wei

    2016-01-01

    The first step in many cellular signaling processes occurs at various types of receptors in the plasma membrane. Membrane cholesterol can alter these signaling pathways of living cells. However, the process in which the interaction of activated receptors is modulated by cholesterol remains unclear. In this study, we measured single-molecule optical trajectories of epidermal growth factor receptors moving in the plasma membranes of two cancerous cell lines and one normal endothelial cell line. A stochastic model was developed and applied to identify critical information from single-molecule trajectories. We discovered that unliganded epidermal growth factor receptors may reside nearby cholesterol-riched regions of the plasma membrane and can move into these lipid domains when subjected to ligand binding. The amount of membrane cholesterol considerably affects the stability of correlated motion of activated epidermal growth factor receptors. Our results provide single-molecule evidence of membrane cholesterol in regulating signaling receptors. Because the three cell lines used for this study are quite diverse, our results may be useful to shed light on the mechanism of cholesterol-mediated interaction between activated receptors in live cells

  2. Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

    Science.gov (United States)

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2015-08-18

    Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

  3. Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

    International Nuclear Information System (INIS)

    Schulster, D.

    1977-01-01

    Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

  4. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    International Nuclear Information System (INIS)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S.; Wada, H.; Horio, Y.

    1990-01-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP PM ) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP PM have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP PM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [ 3 H]oleate but not that of [ 35 S]sulfobromophthalein or [ 14 C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABP PM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP PM and mGOT are closely related

  5. Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

    Science.gov (United States)

    Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

    2014-03-01

    The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. [Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties].

    Science.gov (United States)

    Veklich, T O; Mazur, Iu Iu; Kosterin, S O

    2015-01-01

    Tight control of cytoplasm Ca2+ concentration is essential in cell functioning. Changing of Ca2+ concentration is thorough in smooth muscle cells, because it determines relaxation/constraint process. One of key proteins which control Ca2+ concentration in cytoplasm is Mg2+, ATP-dependent plasma membrane calcium pump. Thus, it is important to find compoumds which allowed one to change Mg2+, ATP-dependent plasma membrane calcium pump activity, as long as this topic is of current interest in biochemical research which regards energy and pharmacomechanical coupling mechanism of muscle excitation and contraction. In this article we generalized literatute and own data about properties of smooth muscle cell plasma membrane Ca(2+)-pump. Stuctural oganization, kinetical properties and molecular biology are considered.

  7. In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

    International Nuclear Information System (INIS)

    Garbarino, J.E.; Hurkman, W.J.; Tanaka, C.K.; DuPont, F.M.

    1991-01-01

    Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ[p 32 P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg 2+ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46 and 28 kilodaltons required millimolar Mg 2+ concentrations and was greatly enhanced by Ca 2+ . When roots of intact plants were labeled with [ 32 P]orthophosphate, polypeptides at approximately 135, 166, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mM NaCl had no effect

  8. Airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

    Energy Technology Data Exchange (ETDEWEB)

    Li, E L.F.; Perdue, J F [Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec, Canada

    1980-10-01

    A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously /sup 125/I- and /sup 32/P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific (/sup 125/I)insulin and (/sup 125/I)thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, and found to be 4 to 5 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 ..mu..g of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating /sup 125/I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.

  9. Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Wendelin Wolf

    Full Text Available The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+ gradient across the plasma membrane generated by the H(+-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.

  10. Fibronectin binding to gangliosides and rat liver plasma membranes

    Energy Technology Data Exchange (ETDEWEB)

    Matyas, G R; Evers, D C; Radinsky, R; Morre, D J

    1986-02-01

    Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (G/sub T1b/ approx. = G/sub D1b/ approx. = G/sub D1a/ > G/sub M1/ >> G/sub M2/ approx. = G/sub D3/ approx. = G/sub M3/) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays. Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides G/sub M1/, G/sub D1a/ or G/sub T1b/ bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent K/sub d/ for binding to mixed rat liver gangliosides of 7.8 x 10/sup -9/ M was determined. This value compared favorably with the apparent K/sub d/ for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 x 10/sup -9/ M for fibronectin modified on the tyrosine residue, or 6.4 x 10/sup -9/ M for fibronectin modified on lysine residues. As shown previously by Grinnell and Minter, fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less (/sup 125/I)fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.

  11. Magnesium Oxide (MgO) pH-sensitive Sensing Membrane in Electrolyte-Insulator-Semiconductor Structures with CF4 Plasma Treatment.

    Science.gov (United States)

    Kao, Chyuan-Haur; Chang, Chia Lung; Su, Wei Ming; Chen, Yu Tzu; Lu, Chien Cheng; Lee, Yu Shan; Hong, Chen Hao; Lin, Chan-Yu; Chen, Hsiang

    2017-08-03

    Magnesium oxide (MgO) sensing membranes in pH-sensitive electrolyte-insulator-semiconductor structures were fabricated on silicon substrate. To optimize the sensing capability of the membrane, CF 4 plasma was incorporated to improve the material quality of MgO films. Multiple material analyses including FESEM, XRD, AFM, and SIMS indicate that plasma treatment might enhance the crystallization and increase the grain size. Therefore, the sensing behaviors in terms of sensitivity, linearity, hysteresis effects, and drift rates might be improved. MgO-based EIS membranes with CF 4 plasma treatment show promise for future industrial biosensing applications.

  12. Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

    Science.gov (United States)

    2015-01-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  13. Adrenal Chromaffin Cells Exposed to 5-ns Pulses Require Higher Electric Fields to Porate Intracellular Membranes than the Plasma Membrane: An Experimental and Modeling Study.

    Science.gov (United States)

    Zaklit, Josette; Craviso, Gale L; Leblanc, Normand; Yang, Lisha; Vernier, P Thomas; Chatterjee, Indira

    2017-10-01

    Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca 2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca 2+ mobilization from Ca 2+ -storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca 2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3-4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

  14. The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs.

    Science.gov (United States)

    Peter, A B; Schittny, J C; Niggli, V; Reuter, H; Sigel, E

    1991-08-01

    Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

  15. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

    DEFF Research Database (Denmark)

    Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

    2008-01-01

    fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

  16. Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: Role in the initiation of sperm motility

    International Nuclear Information System (INIS)

    Gatti, J.L.; Billard, R.; Christen, R.

    1990-01-01

    The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3 H-tetraphenylphosphonium (TPP) and 14 C-thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (less than -40 mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about -15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than -40 mV at pHe 9.0 to -17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+] greater than 20-40 mM or a pH less than 7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement

  17. On the enhancement of pervaporation properties of plasma-deposited hybrid silica membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ngamou, P.H.T.; Creatore, M. [Department of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven (Netherlands); Overbeek, J.P.; Kreiter, R.; Van Veen, H.M.; Vente, J.F. [ECN, Energy research Centre of the Netherlands, Petten (Netherlands); Cuperus, P.F. [SolSep BV, Apeldoorn (Netherlands)

    2013-06-24

    The separation performance of a polymeric-supported hybrid silica membrane in the dehydration process of a butanol-water mixture at 95C has been enhanced by applying a bias to the substrate during the plasma deposition.

  18. A conserved signaling network monitors delivery of sphingolipids to the plasma membrane in budding yeast.

    Science.gov (United States)

    Clarke, Jesse; Dephoure, Noah; Horecka, Ira; Gygi, Steven; Kellogg, Douglas

    2017-10-01

    In budding yeast, cell cycle progression and ribosome biogenesis are dependent on plasma membrane growth, which ensures that events of cell growth are coordinated with each other and with the cell cycle. However, the signals that link the cell cycle and ribosome biogenesis to membrane growth are poorly understood. Here we used proteome-wide mass spectrometry to systematically discover signals associated with membrane growth. The results suggest that membrane trafficking events required for membrane growth generate sphingolipid-dependent signals. A conserved signaling network appears to play an essential role in signaling by responding to delivery of sphingolipids to the plasma membrane. In addition, sphingolipid-dependent signals control phosphorylation of protein kinase C (Pkc1), which plays an essential role in the pathways that link the cell cycle and ribosome biogenesis to membrane growth. Together these discoveries provide new clues as to how growth--dependent signals control cell growth and the cell cycle. © 2017 Clarke et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  19. Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

    Science.gov (United States)

    Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

    2015-07-07

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. H+-ATPase activity from storage tissue of Beta vulgaris. IV. N,N'-dicyclohexylcarbodiimide binding and inhibition of the plasma membrane H+-ATPase

    International Nuclear Information System (INIS)

    Oleski, N.A.; Bennett, A.B.

    1987-01-01

    The molecular weight and isoelectric point of the plasma membrane H + -ATPase from red beet storage tissue were determined using N,N'-dicyclohexylcarbodiimide (DCCD) and a H + -ATPase antibody. When plasma membrane vesicles were incubated with 20 micromolar [ 14 C]-DCCD at 0 0 C, a single 97,000 dalton protein was visualized on a fluorography of a sodium dodecyl sulfate polyacrylamide gel. A close correlation between [ 14 C]DCCD labeling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentration suggests that this 97,000 dalton protein is a component of the plasma membrane H + -ATPase. An antibody raised against the plasma membrane H + -ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two-dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the H + -ATPase to be 6.5

  1. Video Views and Reviews: Golgi Export, Targeting, and Plasma Membrane Caveolae

    Science.gov (United States)

    Watters, Christopher

    2004-01-01

    In this article, the author reviews videos from "Molecular Biology of the Cell (MBC)" depicting various aspects of plasma membrane (PM) dynamics, including the targeting of newly synthesized components and the organization of those PM invaginations called caveolae. The papers accompanying these videos describe, respectively, the constitutive…

  2. Visualization of plasma membrane compartmentalization by high-speed quantum dot tracking

    DEFF Research Database (Denmark)

    Clausen, M. P.; Lagerholm, B. C.

    2013-01-01

    In this study, we have imaged plasma membrane molecules labeled with quantum dots in live cells using a conventional wide-field microscope with high spatial precision at sampling frequencies of 1.75 kHz. Many of the resulting single molecule trajectories are sufficiently long (up to several...

  3. Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

    International Nuclear Information System (INIS)

    Wilcox, C.A.; Olson, E.N.

    1987-01-01

    The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

  4. An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

    Science.gov (United States)

    Bennett, Vann; Lorenzo, Damaris N

    2016-01-01

    Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that

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

    NARCIS (Netherlands)

    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

  6. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  7. New insights into the organization of plasma membrane and its role in signal transduction.

    Science.gov (United States)

    Suzuki, Kenichi G N

    2015-01-01

    Plasma membranes have heterogeneous structures for efficient signal transduction, required to perform cell functions. Recent evidence indicates that the heterogeneous structures are produced by (1) compartmentalization by actin-based membrane skeleton, (2) raft domains, (3) receptor-receptor interactions, and (4) the binding of receptors to cytoskeletal proteins. This chapter provides an overview of recent studies on diffusion, clustering, raft association, actin binding, and signal transduction of membrane receptors, especially glycosylphosphatidylinositol (GPI)-anchored receptors. Studies on diffusion of GPI-anchored receptors suggest that rafts may be small and/or short-lived in plasma membranes. In steady state conditions, GPI-anchored receptors form transient homodimers, which may represent the "standby state" for the stable homodimers and oligomers upon ligation. Furthermore, It is proposed that upon ligation, the binding of GPI-anchored receptor clusters to cytoskeletal actin filaments produces a platform for downstream signaling, and that the pulse-like signaling easily maintains the stability of the overall signaling activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

    Science.gov (United States)

    Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

    2012-06-01

    The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

  9. Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

    Science.gov (United States)

    Halova, Ivana; Draber, Petr

    2016-01-01

    The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

  10. Plasma membrane surface potential: dual effects upon ion uptake and toxicity

    Science.gov (United States)

    Electrical properties of plasma membranes (PMs), partially controlled by the ionic composition of the bathing medium, play significant roles in the distribution of ions at the exterior surface of PMs and in the transport of ions across PMs. The effects of coexistent cations (commonly Al3+, Ca2+, Mg...

  11. Isolation of Synaptosomes, Synaptic Plasma Membranes, and Synaptic Junctional Complexes.

    Science.gov (United States)

    Michaelis, Mary L; Jiang, Lei; Michaelis, Elias K

    2017-01-01

    Isolation of synaptic nerve terminals or synaptosomes provides an opportunity to study the process of neurotransmission at many levels and with a variety of approaches. For example, structural features of the synaptic terminals and the organelles within them, such as synaptic vesicles and mitochondria, have been elucidated with electron microscopy. The postsynaptic membranes are joined to the presynaptic "active zone" of transmitter release through cell adhesion molecules and remain attached throughout the isolation of synaptosomes. These "post synaptic densities" or "PSDs" contain the receptors for the transmitters released from the nerve terminals and can easily be seen with electron microscopy. Biochemical and cell biological studies with synaptosomes have revealed which proteins and lipids are most actively involved in synaptic release of neurotransmitters. The functional properties of the nerve terminals, such as responses to depolarization and the uptake or release of signaling molecules, have also been characterized through the use of fluorescent dyes, tagged transmitters, and transporter substrates. In addition, isolated synaptosomes can serve as the starting material for the isolation of relatively pure synaptic plasma membranes (SPMs) that are devoid of organelles from the internal environment of the nerve terminal, such as mitochondria and synaptic vesicles. The isolated SPMs can reseal and form vesicular structures in which transport of ions such as sodium and calcium, as well as solutes such as neurotransmitters can be studied. The PSDs also remain associated with the presynaptic membranes during isolation of SPM fractions, making it possible to isolate the synaptic junctional complexes (SJCs) devoid of the rest of the plasma membranes of the nerve terminals and postsynaptic membrane components. Isolated SJCs can be used to identify the proteins that constitute this highly specialized region of neurons. In this chapter, we describe the steps involved

  12. Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src

    International Nuclear Information System (INIS)

    Hillsgrove, D.; Shores, C.G.; Parker, J.C.; Maness, P.F.

    1987-01-01

    The authors have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60 v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60 c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [ 35 S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60 c-src . A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. Incubation of the plasma membrane fraction with [ 32 P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicting that the kinase recognized by pp60 c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60 c-src and that this kinase is responsible for band 3 phosphorylation in vitro

  13. LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

    Science.gov (United States)

    Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

    2002-01-01

    The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

  14. Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

    Science.gov (United States)

    Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-07-13

    Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Bright and photostable push-pull pyrene dye visualizes lipid order variation between plasma and intracellular membranes.

    Science.gov (United States)

    Niko, Yosuke; Didier, Pascal; Mely, Yves; Konishi, Gen-ichi; Klymchenko, Andrey S

    2016-01-11

    Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

  16. Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells

    International Nuclear Information System (INIS)

    Sadeghi, H.; Da Silva, A.M.; Klein, C.

    1988-01-01

    The authors describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [ 3 H]myristate, [ 3 H]palmitate, and [ 14 C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [ 14 C]ethanolamine but not with [ 3 H]myristate of [ 3 H]-palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form

  17. Spatial and temporal superresolution concepts to study plasma membrane organization by single molecule fluorescence techniques

    International Nuclear Information System (INIS)

    Ruprecht, V.

    2010-01-01

    Fluorescence microscopy techniques are currently among the most important experimental tools to study cellular processes. Ultra-sensitive detection devices nowadays allow for measuring even individual farnesylacetate labeled target molecules with nanometer spatial accuracy and millisecond time resolution. The emergence of single molecule fluorescence techniques especially contributed to the field of membrane biology and provided basic knowledge on structural and dynamic features of the cellular plasma membrane. However, we are still confronted with a rather fragmentary understanding of the complex architecture and functional interrelations of membrane constituents. In this thesis new concepts in one- and dual-color single molecule fluorescence techniques are presented that allow for addressing organization principles and interaction dynamics in the live cell plasma membrane. Two complementary experimental strategies are described which differ in their detection principle: single molecule fluorescence imaging and fluorescence correlation spectroscopy. The presented methods are discussed in terms of their implementation, accuracy, quantitative and statistical data analysis, as well as live cell applications. State-of-the-art dual color single molecule imaging is introduced as the most direct experimental approach to study interaction dynamics between differently labeled target molecules. New analytical estimates for robust data analysis are presented that facilitate quantitative recording and identification of co localizations in dual color single molecule images. A novel dual color illumination scheme is further described that profoundly extends the current range and sensitivity of conventional dual color single molecule experiments. The method enables working at high surface densities of fluorescent molecules - a feature typically incommensurable with single molecule imaging - and is especially suited for the detection of rare interactions by tracking co localized

  18. Magnetically enhanced triode etching of large area silicon membranes in a molecular bromine plasma

    International Nuclear Information System (INIS)

    Wolfe, J.C.; Sen, S.; Pendharkar, S.V.; Mauger, P.; Shimkunas, A.R.

    1992-01-01

    The optimization of a process for etching 125 mm silicon membranes formed on 150 mm wafers and bonded to Pyrex rings is discussed. A magnetically enhanced triode etching system was designed to provide an intense, remote plasma surrounding the membrane while, at the same time, suppressing the discharge over the membrane itself. For the optimized molecular bromine process, the silicon etch rate is 40 nm/min and the selectivity relative to SiO 2 is 160:1. 14 refs., 6 figs

  19. Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

    Directory of Open Access Journals (Sweden)

    Blachutzik Jörg O

    2012-08-01

    Full Text Available Abstract Background Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana. Results Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan. Conclusion Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

  20. Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity

    NARCIS (Netherlands)

    Golachowska, Magdalena R.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

    2010-01-01

    Recycling endosomes have taken central stage in the intracellular sorting and polarized trafficking of apical and basolateral plasma membrane components. Molecular players in the underlying mechanisms are now emerging, including small GTPases, class V myosins and adaptor proteins. In particular,

  1. From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

    Science.gov (United States)

    Farinha, Carlos M; Canato, Sara

    2017-01-01

    CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

  2. Nutrient Sensing at the Plasma Membrane of Fungal Cells.

    Science.gov (United States)

    Van Dijck, Patrick; Brown, Neil Andrew; Goldman, Gustavo H; Rutherford, Julian; Xue, Chaoyang; Van Zeebroeck, Griet

    2017-03-01

    To respond to the changing environment, cells must be able to sense external conditions. This is important for many processes including growth, mating, the expression of virulence factors, and several other regulatory effects. Nutrient sensing at the plasma membrane is mediated by different classes of membrane proteins that activate downstream signaling pathways: nontransporting receptors, transceptors, classical and nonclassical G-protein-coupled receptors, and the newly defined extracellular mucin receptors. Nontransporting receptors have the same structure as transport proteins, but have lost the capacity to transport while gaining a receptor function. Transceptors are transporters that also function as a receptor, because they can rapidly activate downstream signaling pathways. In this review, we focus on these four types of fungal membrane proteins. We mainly discuss the sensing mechanisms relating to sugars, ammonium, and amino acids. Mechanisms for other nutrients, such as phosphate and sulfate, are discussed briefly. Because the model yeast Saccharomyces cerevisiae has been the most studied, especially regarding these nutrient-sensing systems, each subsection will commence with what is known in this species.

  3. [Ketoconazole and clindamycin efficacy vs oral clindamycin in premature membranes rupture prevention].

    Science.gov (United States)

    Castillo Huerta, Eugenio; Garibay Valencia, Miguel; Mirabent-González, Felio

    2008-07-01

    Vulvovaginitis is one of the main causes of premature membrane rupture. To evaluate effectiveness of a combination of ketoconazole (400 mg) and clindamycin (100 mg) in vaginal tablets, compared with clindamicyn alone (600 mg/daily) orally, for six days, to prevent premature membrane rupture in patients with vulvovaginitis. Longitudinal, prospective, comparative, randomized, double-blind, double-dummy study in patients older than 18 years, during them third trimester of normoevolutive pregnancy with symptomatic vulvovaginitis. Patients were monitored as out patient. Genital secretion culture and fresh studies were made. Signs and symptoms were evaluated in regular intervals: 4, 7 and 11 days. Pregnancy control was performed every three weeks, until childbirth or premature membrane rupture. 105 patients were included: 53 in the group of ketoconazole and clindamicyn (1), and 52 in the group of clindamycin alone (2). Symptoms were similar in both groups of treatment, without statistically significant differences. A case of group 2 has premature membrane rupture (p = 0.495). C. albicans was cultured in 35% of group 2 and in 11% of group 2. No adverse events with treatments were reported. The combination of ketoconazole and cindamycin was effective to prevent premature membrane rupture in patients with vulvovaginitis.

  4. Potassium as an intrinsic uncoupler of the plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Palmgren, Michael Gjedde; Buch-Pedersen, Morten Jeppe

    The plant plasma membrane proton pump (H(+)-ATPase) is stimulated by potassium, but it has remained unclear whether potassium is actually transported by the pump or whether it serves other roles. We now show that K(+) is bound to the proton pump at a site involving Asp(617) in the cytoplasmic...

  5. Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture

    International Nuclear Information System (INIS)

    Gerton, G.L.; Millette, C.F.

    1986-01-01

    Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [ 35 S]methionine or [ 3 H] fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells

  6. Plasma Membrane H(+)-ATPase Regulation in the Center of Plant Physiology.

    Science.gov (United States)

    Falhof, Janus; Pedersen, Jesper Torbøl; Fuglsang, Anja Thoe; Palmgren, Michael

    2016-03-07

    The plasma membrane (PM) H(+)-ATPase is an important ion pump in the plant cell membrane. By extruding protons from the cell and generating a membrane potential, this pump energizes the PM, which is a prerequisite for growth. Modification of the autoinhibitory terminal domains activates PM H(+)-ATPase activity, and on this basis it has been hypothesized that these regulatory termini are targets for physiological factors that activate or inhibit proton pumping. In this review, we focus on the posttranslational regulation of the PM H(+)-ATPase and place regulation of the pump in an evolutionary and physiological context. The emerging picture is that multiple signals regulating plant growth interfere with the posttranslational regulation of the PM H(+)-ATPase. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  7. Comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane between normal and preeclampsia pregnancies with high-resolution mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Fuqiang Wang

    Full Text Available Preeclampsia is a serious complication of pregnancy, which affects 2-8% of all pregnancies and is one of the leading causes of maternal and perinatal mortality and morbidity worldwide. To better understand the molecular mechanisms involved in pathological development of placenta in preeclampsia, we used high-resolution LC-MS/MS technologies to construct a comparative N-glycoproteomic and phosphoproteomic profiling of human placental plasma membrane in normal and preeclamptic pregnancies. A total of 1027 N-glyco- and 2094 phospho- sites were detected in human placental plasma membrane, and 5 N-glyco- and 38 phospho- proteins, respectively, with differentially expression were definitively identified between control and preeclamptic placental plasma membrane. Further bioinformatics analysis indicated that these differentially expressed proteins correlate with several specific cellular processes occurring during pathological changes of preeclamptic placental plasma membrane.

  8. C_1_8-attached membrane funnel-based spray ionization mass spectrometry for quantification of anti-diabetic drug from human plasma

    International Nuclear Information System (INIS)

    Li, Wan; Chen, Xiangfeng; Wong, Y.-L. Elaine; Hung, Y.-L. Winnie; Wang, Ze; Deng, Liulin; Dominic Chan, T.-W.

    2016-01-01

    In this work, sorbent-attached membrane funnel-based spray ionization mass spectrometry was explored for quantitative analysis of anti-diabetic drugs spiked in human plasma. C_1_8-attached membrane funnel was fabricated for in situ extraction and clean-up to alleviate matrix suppression effect in the ionization process. Repaglinide was used as a target analyte of anti-diabetic drugs. Under optimal working conditions, good linearity (R"2 > 0.99) was obtained in the concentration range of 1–100 ng mL"−"1. The method detection limit of target drugs spiked in the human plasma was around 0.30 ng mL"−"1. Through the application of an isotope-labeled internal standard, the signal fluctuation caused by residual background matrices was largely alleviated and the precision of measurement (RSD) was below 15%. The recovery of repaglinide for 5, 25, and 100 ng mL"−"1 of spiked human plasma matrixes ranged from 87% to 112%. The developed method was successfully applied to determine repaglinide in plasma volunteers who orally received a dose of drug association. Our results demonstrated that membrane funnel-based spray is a simple and sensitive method for rapid screening analysis of complex biological samples. - Highlights: • Sorbent attached membrane funnel based spray platform was used for drug determination in human plasma. • The matrix suppression effect of human plasma was largely eliminated. • The method was applied to determine repaglinide in plasma volunteers. • Membrane funnel-based spray is promising for analysis of biological samples.

  9. Quantitative transporter proteomics by liquid chromatography with tandem mass spectrometry: addressing methodologic issues of plasma membrane isolation and expression-activity relationship.

    Science.gov (United States)

    Kumar, Vineet; Prasad, Bhagwat; Patilea, Gabriela; Gupta, Anshul; Salphati, Laurent; Evers, Raymond; Hop, Cornelis E C A; Unadkat, Jashvant D

    2015-02-01

    To predict transporter-mediated drug disposition using physiologically based pharmacokinetic models, one approach is to measure transport activity and relate it to protein expression levels in cell lines (overexpressing the transporter) and then scale these to via in vitro to in vivo extrapolation (IVIVE). This approach makes two major assumptions. First, that the expression of the transporter is predominantly in the plasma membrane. Second, that there is a linear correlation between expression level and activity of the transporter protein. The present study was conducted to test these two assumptions. We evaluated two commercially available kits that claimed to separate plasma membrane from other cell membranes. The Qiagen Qproteome kit yielded very little protein in the fraction purported to be the plasma membrane. The Abcam Phase Separation kit enriched the plasma membrane but did not separate it from other intracellular membranes. For the Abcam method, the expression level of organic anion-transporting polypeptides (OATP) 1B1/2B1 and breast cancer resistance protein (BCRP) proteins in all subcellular fractions isolated from cells or human liver tissue tracked that of Na⁺-K⁺ ATPase. Assuming that Na⁺-K⁺ ATPase is predominantly located in the plasma membrane, these data suggest that the transporters measured are also primarily located in the plasma membrane. Using short hairpin RNA, we created clones of cell lines with varying degrees of OATP1B1 or BCRP expression level. In these clones, transport activity of OATP1B1 or BCRP was highly correlated with protein expression level (r² > 0.9). These data support the use of transporter expression level data and activity data from transporter overexpressing cell lines for IVIVE of transporter-mediated disposition of drugs. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  10. Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts. [Annual report], May 16, 1993--January 29, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Steponkus, P.L.

    1994-06-01

    Our aim is to provide a mechanistic understanding of the cellular and molecular aspects of freezing injury and cold acclimation from a perspective of the structural and functional integrity of the plasma membrane-the primary site of freezing injury in winter cereals. We established that destabilization of the plasma membrane of winter rye, the most freezing-tolerant winter cereal, can result from several different lesions: expansion induced lysis, lamellar-to-hexagonal II phase transitions, and the fracture-jump lesion. The occurrence and incidence of these various lesions, depends on the freeze/thaw protocol and the stage of cold acclimation. In non-acclimated leaves and protoplasts, expansion-induced lysis is the predominant lesion at temperatures between {minus}2 and {minus}5{degree}C, whereas freeze-induced formation of the H{sub II} phase is the predominant lesion at temperatures below {minus}10{degree}C. We investigated whether the difference in freezing tolerance and the threshold temperatures at which the lesions occur in rye and oat are a consequence of differences in the lipid composition of the plasma membrane. There are substantial differences between rye and oat cell membranes both before and after cold acclimation. The plasma membrane of oat contains greater proportions of acylated sterylglucosides and cerebrosides than that of rye, and there is little change in these two lipid classes during cold acclimation. The lyotropic phase behavior of lipid mixtures that resemble the plasma membrane of rye and oat was studied. The differences in lipid composition of rye and oat are of mechanistic significance because of their influence on the hydration characteristics of the plasma membrane, the propensity for dehydration-induced lipid-lipid demixing, and the intrinsic curvature of the lipid monolayers. These studies suggest that strategies for improving the freezing tolerance of winter cereals should include approaches to modify membrane lipid composition.

  11. Antifouling coatings via plasma polymerization and atom transfer radical polymerization on thin film composite membranes for reverse osmosis

    Science.gov (United States)

    Hirsch, Ulrike; Ruehl, Marco; Teuscher, Nico; Heilmann, Andreas

    2018-04-01

    A major drawback to otherwise highly efficient membrane-based desalination techniques like reverse osmosis (RO) is the susceptibility of the membranes to biofouling. In this work, a combination of plasma activation, plasma bromination and surface-initiated atom transfer radical polymerization (si-ATRP) of hydrophilic and zwitterionic monomers, namely hydroxyethyl methacrylate (HEMA), 2-methacryloyloxyethyl phosphorylcholine (MPC) and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)ammonium hydroxide (SBMA), was applied to generate non-specific, anti-adhesive coatings on thin film composite (TFC) membranes. The antifouling effect of the coatings was shown by short-time batch as well as long-time steady state cultivation experiments with the microorganism Pseudomonas fluorescens. It could be shown that plasma functionalization and polymerization is possible on delicate thin film composite membranes without restricting their filtration performance. All modified membranes showed an increased resistance towards the adhesion of Pseudomonas fluorescens. On average, the biofilm coverage was reduced by 51.4-12.6% (for HEMA, SBMA, and MPC), the highest reduction was monitored for MPC with a biofilm reduction by 85.4%. The hydrophilic coatings applied did not only suppress the adhesion of Pseudomonas fluorescens, but also significantly increase the permeate flux of the membranes relative to uncoated membranes. The stability of the coatings was however not ideal and will have to be improved for future commercial use.

  12. Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition

    Directory of Open Access Journals (Sweden)

    Antônio Jesus Dorighetto Cogo

    2018-02-01

    Full Text Available Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

  13. Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

    Science.gov (United States)

    Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

    2014-01-01

    Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

  14. Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

    Science.gov (United States)

    Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

    2011-10-01

    The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

  15. In Vitro Comparative Study of Oxygen Plasma Treated Poly(Lactic⁻Co⁻Glycolic) (PLGA) Membranes and Supported Nanostructured Oxides for Guided Bone Regeneration Processes.

    Science.gov (United States)

    Torres-Lagares, Daniel; Castellanos-Cosano, Lizett; Serrera-Figallo, Maria-Angeles; López-Santos, Carmen; Barranco, Angel; Rodríguez-González-Elipe, Agustín; Gutierrez-Perez, Jose-Luis

    2018-05-08

    (1) Background: The use of physical barriers to prevent the invasion of gingival and connective tissue cells into bone cavities during the healing process is called guided bone regeneration. The objective of this in-vitro study was to compare the growth of human osteoblasts on Poly(Lactic⁻co⁻Glycolic) (PLGA) membranes modified with oxygen plasma and Hydroxyapatite (HA), silicon dioxide (SiO₂), and titanium dioxide (TiO₂) composite nanoparticles, respectively. (2) Methods: All the membranes received a common treatment with oxygen plasma and were subsequently treated with HA nanostructured coatings (n = 10), SiO₂ (n = 10) and TiO₂ (n = 10), respectively and a PLGA control membrane (n = 10). The assays were performed using the human osteoblast line MG-63 acquired from the Center for Scientific Instrumentation (CIC) from the University of Granada. The cell adhesion and the viability of the osteoblasts were analyzed by means of light-field microphotographs of each condition with the inverted microscope Axio Observer A1 (Carl Zeiss). For the determination of the mitochondrial energy balance, the MitoProbe™ JC-1 Assay Kit was employed. For the determination of cell growth and the morphology of adherent osteoblasts, two techniques were employed: staining with phalloidin-TRITC and staining with DAPI. (3) Results: The modified membranes that show osteoblasts with a morphology more similar to the control osteoblasts follow the order: PLGA/PO₂/HA > PLGA/PO₂/SiO₂ > PLGA/PO₂/TiO₂ > PLGA ( p membranes was observed as follows: PLGA/PO₂/SiO₂ > PLGA/PO₂/HA > PLGA/PO₂/TiO₂ > PLGA ( p membranes PLGA/PO₂/HA and PLGA/PO₂/SiO₂. (4) Conclusion: The membrane in which osteoblasts show characteristics more similar to the control osteoblasts is the PLGA/PO₂/HA, followed by the PLGA/PO₂/SiO₂.

  16. Lectin receptor kinase LecRK-b2 localizes to plasma membrane and ...

    African Journals Online (AJOL)

    -b2, has been characterized. Confocal microscopy images showed that the LecRK-b2-GFP fusion protein is localized to plasma membrane. The results of yeast 2 hybrid showed that lectin domain of LecRK-b2 had selfinteraction, while the ...

  17. Effect of colchicine on rat small intestinal absorptive cells. II. Distribution of label after incorporation of [3H]fucose into plasma membrane glycoproteins

    International Nuclear Information System (INIS)

    Ellinger, A.; Pavelka, M.; Gangl, A.

    1983-01-01

    By means of radioautography the influence was tested of various periods (5, 15, 30, 40 min, 2 hr) of pretreatment with colchicine, administered intraperitoneally to rats at a dosage of 0.5 mg/100 g of body weight, on the intracellular pathway of [ 3 H]fucose in absorptive cells of the small intestine. Administration of colchicine for 30 min and longer time intervals causes delay in the insertion of [ 3 H]fucose into the oligosaccharide chains of glycoconjugates in the Golgi apparatus, and results in redistribution of the label apparent over the different portions of the plasma membrane. In controls, at 2 and 4 hr after administration of [ 3 H]fucose the apical plasma membrane is strongly labeled. Colchicine causes equalization of the reaction of apical and basolateral regions of the plasma membrane: the number of silver grains attributable to the apical plasma membrane is reduced; following treatment with colchicine, apical portions of the plasma membrane comprise 31.6 +/- 1.8% of the silver grains, 38.6 +/- 3.8% are attributable to basolateral membrane regions. The colchicine-induced equalization of the density of label of apical and basolateral regions of the plasma membrane, in addition to the occurrence of basolateral microvillus borders, suggests microtubules to be important in the maintenance of the polar organization of small intestinal absorptive cells

  18. Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

    International Nuclear Information System (INIS)

    Kasai, M.; Muto, S.

    1990-01-01

    Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca2+ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment. 45Ca2+ transport was assayed by membrane filtration technique. Results showed that Ca2+ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca2+ transport system had a high affinity for Ca2+(Km(Ca2+) = 0.4 microM) and ATP(Km(ATP) = 3.9 microM), and showed pH optimum at 7.5. ATP-dependent Ca2+ uptake in the plasma membrane vesicles was stimulated in the presence of Cl- or NO3-. Quenching of quinacrine fluorescence showed that these anions also induced H+ transport into the vesicles. The Ca2+ uptake stimulated by Cl- was dependent on the activity of H+ transport into the vesicles. However, carbonylcyanide m-chlorophenylhydrazone (CCCP) and VO4(3-) which is known to inhibit the H+ pump associated with the plasma membrane, canceled almost all of the Cl(-)-stimulated Ca2+ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca2+ uptake into the vesicles. These results suggest that the Cl(-)-stimulated Ca2+ uptake is caused by the efflux of H+ from the vesicles by the operation of Ca2+/H+ antiport system in the plasma membrane. In Cl(-)-free medium, H+ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca2+ uptake into the vesicles. These results suggest that two Ca2+ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca2+ transport system (Ca2+ pump) and the other is a Ca2+/H+ antiport system. Little difference in characteristics of Ca2+ transport was observed between the plasma membranes isolated from etiolated and green corn leaves

  19. Modification of plasma membrane electron transport in cultured rose cells by UV-C radiation and fungal elicitor

    International Nuclear Information System (INIS)

    Murphy, T.M.; Auh, C.K.; Schorr, R.; Grobe, C.

    1991-01-01

    Previous experiments have shown that treatments of suspension-cultured cells of Rosa damascena Mill. with UV radiation or with fungal elicitors stimulates the synthesis of H 2 O 2 by the cells. To test the hypothesis that this synthesis involves reduction of O 2 at the plasma membrane and to identify the mechanism of the reduction, we have determined the effects of UV and elicitor on redox reactions associated with the plasma membrane. Elicitor prepared from cell walls of Phytophthora sp. (14 μg solids/ml) inhibited the reduction of ferricyanide by intact cells by 98%; UV-C (primarily 254 nm, up to 19,500 J/m 2 ) inhibited this reduction by 40%. Neither treatment inhibited the reduction of Fe(III)-EDTA by intact cells. Intact cells oxidized NADH in the absence of external oxidizing agent, and the rate of oxidation was increased by UV and elicitor. Cells that were poisoned with arsenite and CCCP catalyzed the reduction of Fe(III)-EDTA in the presence of external NADH, and this ability was slightly stimulated by UV and elicitor. UV irradiation (6,480 J/m 2 ) of cells resulted in a 27% inhibition of the specific activity of NADH-ferricyanide oxidoreductase in plasma membrane isolated from those cells. Elicitor treatment of cells for at least 90 min resulted in a 50% inhibition of the enzyme's specific activity in isolated plasma membrane; this inhibition was reversed by addition of Triton-X100 in the assay mixture. The results suggest that UV and elicitor alter the flow of electrons in the plasma membrane, reversibly inhibiting NADH-cytochrome b reductase, the putative key enzyme in the pathway of ferricyanide reduction, and stimulating (or at least not inhibiting) the pathway of Fe(III)-EDTA reduction

  20. Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin

    International Nuclear Information System (INIS)

    Ponec, M.; Kempenaar, J.; Weerheim, A.; Boonstra, J.

    1987-01-01

    We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14 C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression

  1. Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase1[OPEN

    Science.gov (United States)

    Tan, Li Xuan; Bushey, Daniel B.; Swanson, Sarah J.

    2018-01-01

    A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase. PMID:29042459

  2. Autoinhibitory Regulation of Plasma Membrane H+-ATPases

    DEFF Research Database (Denmark)

    Pedersen, Jesper Torbøl

    Electrochemical gradients across cell membranes are essential for nutrient uptake. In plant and fungal cells the electrochemical gradient across the plasma membrane (PM) can build much higher than in mammalian cells. The protein responsible for this gradient is the essential PM H+-ATPase that uses...... resolution 3D structure the mechanism behind is only poorly understood. This thesis aimed at illuminating the autoinhibitory mechanism in plant and yeast PM H+-ATPases and below some of our main findings will be highlighted. The two terminal domains of the PM H+-ATPases have several amino acid residues...... that can be phosphorylated, and it has been demonstrated that these phosphorylation sites in both plant and yeast are highly involved in the regulation of terminal autoinhibition. In this study we used a phylogenetic analysis to investigate the evolutionary development of these phosphorylation sites...

  3. C{sub 18}-attached membrane funnel-based spray ionization mass spectrometry for quantification of anti-diabetic drug from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wan [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Chen, Xiangfeng, E-mail: xiangfchensdas@163.com [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Shandong Analysis and Test Centre, Shandong Academy of Sciences, Jinan, Shandong (China); Wong, Y.-L. Elaine; Hung, Y.-L. Winnie; Wang, Ze; Deng, Liulin [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Dominic Chan, T.-W., E-mail: twdchan@cuhk.edu.hk [Department of Chemistry, The Chinese University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong)

    2016-08-24

    In this work, sorbent-attached membrane funnel-based spray ionization mass spectrometry was explored for quantitative analysis of anti-diabetic drugs spiked in human plasma. C{sub 18}-attached membrane funnel was fabricated for in situ extraction and clean-up to alleviate matrix suppression effect in the ionization process. Repaglinide was used as a target analyte of anti-diabetic drugs. Under optimal working conditions, good linearity (R{sup 2} > 0.99) was obtained in the concentration range of 1–100 ng mL{sup −1}. The method detection limit of target drugs spiked in the human plasma was around 0.30 ng mL{sup −1}. Through the application of an isotope-labeled internal standard, the signal fluctuation caused by residual background matrices was largely alleviated and the precision of measurement (RSD) was below 15%. The recovery of repaglinide for 5, 25, and 100 ng mL{sup −1} of spiked human plasma matrixes ranged from 87% to 112%. The developed method was successfully applied to determine repaglinide in plasma volunteers who orally received a dose of drug association. Our results demonstrated that membrane funnel-based spray is a simple and sensitive method for rapid screening analysis of complex biological samples. - Highlights: • Sorbent attached membrane funnel based spray platform was used for drug determination in human plasma. • The matrix suppression effect of human plasma was largely eliminated. • The method was applied to determine repaglinide in plasma volunteers. • Membrane funnel-based spray is promising for analysis of biological samples.

  4. Proton-sensing transistor systems for detecting ion leakage from plasma membranes under chemical stimuli.

    Science.gov (United States)

    Imaizumi, Yuki; Goda, Tatsuro; Schaffhauser, Daniel F; Okada, Jun-Ichi; Matsumoto, Akira; Miyahara, Yuji

    2017-03-01

    The membrane integrity of live cells is routinely evaluated for cytotoxicity induced by chemical or physical stimuli. Recent progress in bioengineering means that high-quality toxicity validation is required. Here, we report a pH-sensitive transistor system developed for the continuous monitoring of ion leakage from cell membranes upon challenge by toxic compounds. Temporal changes in pH were generated with high reproducibility via periodic flushing of HepG2 cells on a gate insulator of a proton-sensitive field-effect transistor with isotonic buffer solutions with/without NH 4 Cl. The pH transients at the point of NH 4 Cl addition/withdrawal originated from the free permeation of NH 3 across the semi-permeable plasma membranes, and the proton sponge effect produced by the ammonia equilibrium. Irreversible attenuation of the pH transient was observed when the cells were subjected to a membrane-toxic reagent. Experiments and simulations proved that the decrease in the pH transient was proportional to the area of the ion-permeable pores on the damaged plasma membranes. The pH signal was correlated with the degree of hemolysis produced by the model reagents. The pH assay was sensitive to the formation of molecularly sized pores that were otherwise not measurable via detection of the leakage of hemoglobin, because the hydrodynamic radius of hemoglobin was greater than 3.1nm in the hemolysis assay. The pH transient was not disturbed by inherent ion-transporter activity. The ISFET assay was applied to a wide variety of cell types. The system presented here is fast, sensitive, practical and scalable, and will be useful for validating cytotoxins and nanomaterials. The plasma membrane toxicity and hemolysis are widely and routinely evaluated in biomaterials science and biomedical engineering. Despite the recent development of a variety of methods/materials for efficient gene/drug delivery systems to the cytosol, the methodologies for safety validation remain unchanged in

  5. Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

    International Nuclear Information System (INIS)

    Bode, D.C.; Molinoff, P.B.

    1988-01-01

    The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The β-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of β-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 0 C was not associated with a change in anisotropy

  6. The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.

    Science.gov (United States)

    Fernandes, Kelly Souza; de Souza, Paulo Eduardo Narcizo; Dorta, Miriam Leandro; Alonso, Antonio

    2017-01-01

    In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (c w50 ) and the cell membrane (c m50 ) and the membrane-aqueous medium partition coefficient of MT (K). With a c w50 of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had c w50 values of 2.4-3.1μM. The estimated c m50 of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Aquatic biofouling prevention by electrically charged nanocomposite polymer thin film membranes.

    Science.gov (United States)

    de Lannoy, Charles-François; Jassby, David; Gloe, Katie; Gordon, Alexander D; Wiesner, Mark R

    2013-03-19

    Electrically conductive polymer-nanocomposite (ECPNC) tight nanofiltration (NF) thin film membranes were demonstrated to have biofilm-preventing capabilities under extreme bacteria and organic material loadings. A simple route to the creation and application of these polyamide-carbon nanotube thin films is also reported. These thin films were characterized with SEM and TEM as wel