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Sample records for arabidopsis plasma membrane

  1. Advancements in the analysis of the Arabidopsis plasma membrane proteome

    Directory of Open Access Journals (Sweden)

    Koste eYadeta

    2013-04-01

    Full Text Available The plasma membrane (PM regulates diverse processes essential to plant growth, development, and survival in an ever-changing environment. In addition to maintaining normal cellular homeostasis and plant nutrient status, PM proteins perceive and respond to a myriad of environmental cues. Here we review recent advances in the analysis of the plant PM proteome with a focus on the model plant Arabidopsis thaliana. Due to membrane heterogeneity, hydrophobicity, and low relative abundance, analysis of the PM proteome has been a special challenge. Various experimental techniques to enrich PM proteins and different protein and peptide separation strategies have facilitated the identification of thousands of integral and membrane-associated proteins. Numerous classes of proteins are present at the PM with diverse biological functions. Plasma membrane microdomains have attracted much attention. However, it still remains a challenge to characterize these cell membrane compartments. Dynamic changes in the PM proteome in response to different biotic and abiotic stimuli are highlighted. Future prospects for PM proteomics research are also discussed.

  2. 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...

  3. The effect of a genetically reduced plasma membrane protonmotive force on vegetative growth of Arabidopsis.

    Science.gov (United States)

    Haruta, Miyoshi; Sussman, Michael R

    2012-03-01

    The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H(+)-ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.

  4. ACA12 Is a Deregulated Isoform of Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana

    Science.gov (United States)

    Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F.; De Michelis, Maria Ida

    2014-01-01

    Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues - highly conserved in other ACA isoforms - localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation. PMID:24101142

  5. Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

    Science.gov (United States)

    Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

    1999-01-01

    Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

  6. Plasma Membrane Proteomics of Arabidopsis Suspension-Cultured Cells Associated with Growth Phase Using Nano-LC-MS/MS.

    Science.gov (United States)

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

    2018-01-01

    Arabidopsis thaliana suspension-cultured cells (T87 line) are important model system for studies of responses to biotic and abiotic stresses at the cellular level in vitro since the cells have certain advantages compared with the whole plant system. However, the physiological and morphological characteristics of the cells are influenced by the progress of the growth phase of cells, which may result in different stress tolerance. To obtain comprehensive proteome profiles of the plasma membrane of Arabidopsis thaliana T87 suspension-cultured cells at the lag, log, or stationary growth phase, a shotgun proteomics method using nano-LC-MS/MS is used. The results obtained indicate that proteome profiles of the plasma membrane with the progress of the growth phase of cells dynamically changed, which may be associated with the physiological and morphological characteristics of the plasma membrane of the suspension-cultured cells. The proteomics results are further applied to explain different responsive patterns in the plasma membrane to cold acclimation and ABA treatment, which lead to understanding of different freezing tolerance associated with the growth phase of the cells.

  7. Simplified Enrichment of Plasma Membrane Proteins from Arabidopsis thaliana Seedlings Using Differential Centrifugation and Brij-58 Treatment.

    Science.gov (United States)

    Collins, Carina A; Leslie, Michelle E; Peck, Scott C; Heese, Antje

    2017-01-01

    The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

  8. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants

    Science.gov (United States)

    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-01-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na+/H+) antiporter that transports Na+ into the vacuole and exports H+ into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na+/H+ antiporter that exports Na+ to the extracellular space and imports H+ into the plant cell. Plants rely on these enzymes either to keep Na+ out of the cell or to sequester Na+ into vacuoles to avoid the toxic level of Na+ in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. PMID:26985021

  9. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants.

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    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-05-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na(+)/H(+)) antiporter that transports Na(+) into the vacuole and exports H(+) into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na(+)/H(+) antiporter that exports Na(+) to the extracellular space and imports H(+) into the plant cell. Plants rely on these enzymes either to keep Na(+) out of the cell or to sequester Na(+) into vacuoles to avoid the toxic level of Na(+) in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  10. CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

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    Lei, Lei; Singh, Abhishek; Bashline, Logan; Li, Shundai; Yingling, Yaroslava G.; Gu, Ying

    2015-10-06

    Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.

  11. Relative abundance of integral plasma membrane proteins in Arabidopsis leaf and root tissue determined by metabolic labeling and mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Katja Bernfur

    Full Text Available Metabolic labeling of proteins with a stable isotope ((15N in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome.

  12. Arabidopsis Novel Glycine-Rich Plasma Membrane PSS1 Protein Enhances Disease Resistance in Transgenic Soybean Plants1[OPEN

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    Wang, Bing; Sumit, Rishi; Srivastava, Subodh K.; Yang, Yang; Swaminathan, Sivakumar

    2018-01-01

    Nonhost resistance is defined as the immunity of a plant species to all nonadapted pathogen species. Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 is nonhost to the oomycete plant pathogen Phytophthora sojae and the fungal plant pathogen Fusarium virguliforme that are pathogenic to soybean (Glycine max). Previously, we reported generating the pss1 mutation in the pen1-1 genetic background as well as genetic mapping and characterization of the Arabidopsis nonhost resistance Phytophthora sojae-susceptible gene locus, PSS1. In this study, we identified six candidate PSS1 genes by comparing single-nucleotide polymorphisms of (1) the bulked DNA sample of seven F2:3 families homozygous for the pss1 allele and (2) the pen1-1 mutant with Columbia-0. Analyses of T-DNA insertion mutants for each of these candidate PSS1 genes identified the At3g59640 gene encoding a glycine-rich protein as the putative PSS1 gene. Later, complementation analysis confirmed the identity of At3g59640 as the PSS1 gene. PSS1 is induced following P. sojae infection as well as expressed in an organ-specific manner. Coexpression analysis of the available transcriptomic data followed by reverse transcriptase-polymerase chain reaction suggested that PSS1 is coregulated with ATG8a (At4g21980), a core gene in autophagy. PSS1 contains a predicted single membrane-spanning domain. Subcellular localization study indicated that it is an integral plasma membrane protein. Sequence analysis suggested that soybean is unlikely to contain a PSS1-like defense function. Following the introduction of PSS1 into the soybean cultivar Williams 82, the transgenic plants exhibited enhanced resistance to F. virguliforme, the pathogen that causes sudden death syndrome. PMID:29101280

  13. Arabidopsis TWISTED DWARF1 functionally interacts with Auxin Exporter ABCB1 on the root plasma membrane

    DEFF Research Database (Denmark)

    Wang, Bangjun; Bailly, Aurélien; Zwiewka, Marta

    2013-01-01

    Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding...... protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological....... In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root...

  14. PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis.

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    Simon, Sibu; Skůpa, Petr; Viaene, Tom; Zwiewka, Marta; Tejos, Ricardo; Klíma, Petr; Čarná, Mária; Rolčík, Jakub; De Rycke, Riet; Moreno, Ignacio; Dobrev, Petre I; Orellana, Ariel; Zažímalová, Eva; Friml, Jiří

    2016-07-01

    Plant development mediated by the phytohormone auxin depends on tightly controlled cellular auxin levels at its target tissue that are largely established by intercellular and intracellular auxin transport mediated by PIN auxin transporters. Among the eight members of the Arabidopsis PIN family, PIN6 is the least characterized candidate. In this study we generated functional, fluorescent protein-tagged PIN6 proteins and performed comprehensive analysis of their subcellular localization and also performed a detailed functional characterization of PIN6 and its developmental roles. The localization study of PIN6 revealed a dual localization at the plasma membrane (PM) and endoplasmic reticulum (ER). Transport and metabolic profiling assays in cultured cells and Arabidopsis strongly suggest that PIN6 mediates both auxin transport across the PM and intracellular auxin homeostasis, including the regulation of free auxin and auxin conjugates levels. As evidenced by the loss- and gain-of-function analysis, the complex function of PIN6 in auxin transport and homeostasis is required for auxin distribution during lateral and adventitious root organogenesis and for progression of these developmental processes. These results illustrate a unique position of PIN6 within the family of PIN auxin transporters and further add complexity to the developmentally crucial process of auxin transport. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  15. ACA12 is a deregulated isoform of plasma membrane Ca²⁺-ATPase of Arabidopsis thaliana.

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    Limonta, Margherita; Romanowsky, Shawn; Olivari, Claudio; Bonza, Maria Cristina; Luoni, Laura; Rosenberg, Alexa; Harper, Jeffrey F; De Michelis, Maria Ida

    2014-03-01

    Plant auto-inhibited Ca²⁺-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsis thaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca²⁺-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues--highly conserved in other ACA isoforms--localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.

  16. Bipolar Plasma Membrane Distribution of Phosphoinositides and Their Requirement for Auxin-Mediated Cell Polarity and Patterning in Arabidopsis.

    Science.gov (United States)

    Tejos, Ricardo; Sauer, Michael; Vanneste, Steffen; Palacios-Gomez, Miriam; Li, Hongjiang; Heilmann, Mareike; van Wijk, Ringo; Vermeer, Joop E M; Heilmann, Ingo; Munnik, Teun; Friml, Jiří

    2014-05-01

    Cell polarity manifested by asymmetric distribution of cargoes, such as receptors and transporters, within the plasma membrane (PM) is crucial for essential functions in multicellular organisms. In plants, cell polarity (re)establishment is intimately linked to patterning processes. Despite the importance of cell polarity, its underlying mechanisms are still largely unknown, including the definition and distinctiveness of the polar domains within the PM. Here, we show in Arabidopsis thaliana that the signaling membrane components, the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] as well as PtdIns4P 5-kinases mediating their interconversion, are specifically enriched at apical and basal polar plasma membrane domains. The PtdIns4P 5-kinases PIP5K1 and PIP5K2 are redundantly required for polar localization of specifically apical and basal cargoes, such as PIN-FORMED transporters for the plant hormone auxin. As a consequence of the polarity defects, instructive auxin gradients as well as embryonic and postembryonic patterning are severely compromised. Furthermore, auxin itself regulates PIP5K transcription and PtdIns4P and PtdIns(4,5)P 2 levels, in particular their association with polar PM domains. Our results provide insight into the polar domain-delineating mechanisms in plant cells that depend on apical and basal distribution of membrane lipids and are essential for embryonic and postembryonic patterning. © 2014 American Society of Plant Biologists. All rights reserved.

  17. An early nodulin-like protein accumulates in the sieve element plasma membrane of Arabidopsis

    DEFF Research Database (Denmark)

    Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.

    2007-01-01

    Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicacea...

  18. Bipolar Plasma Membrane Distribution of Phosphoinositides and Their Requirement for Auxin-Mediated Cell Polarity and Patterning in Arabidopsis[W

    Science.gov (United States)

    Tejos, Ricardo; Sauer, Michael; Vanneste, Steffen; Palacios-Gomez, Miriam; Li, Hongjiang; Heilmann, Mareike; van Wijk, Ringo; Vermeer, Joop E.M.; Heilmann, Ingo; Munnik, Teun; Friml, Jiří

    2014-01-01

    Cell polarity manifested by asymmetric distribution of cargoes, such as receptors and transporters, within the plasma membrane (PM) is crucial for essential functions in multicellular organisms. In plants, cell polarity (re)establishment is intimately linked to patterning processes. Despite the importance of cell polarity, its underlying mechanisms are still largely unknown, including the definition and distinctiveness of the polar domains within the PM. Here, we show in Arabidopsis thaliana that the signaling membrane components, the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as well as PtdIns4P 5-kinases mediating their interconversion, are specifically enriched at apical and basal polar plasma membrane domains. The PtdIns4P 5-kinases PIP5K1 and PIP5K2 are redundantly required for polar localization of specifically apical and basal cargoes, such as PIN-FORMED transporters for the plant hormone auxin. As a consequence of the polarity defects, instructive auxin gradients as well as embryonic and postembryonic patterning are severely compromised. Furthermore, auxin itself regulates PIP5K transcription and PtdIns4P and PtdIns(4,5)P2 levels, in particular their association with polar PM domains. Our results provide insight into the polar domain–delineating mechanisms in plant cells that depend on apical and basal distribution of membrane lipids and are essential for embryonic and postembryonic patterning. PMID:24876254

  19. CHX14 is a plasma membrane K-efflux transporter that regulates K+ redistribution in "Arabidopsis thaliana"

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    Potassium (K(+)) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. "CHX14" expression was induced by elevated K(+) and histochemical analysis...

  20. Bipolar Plasma Membrane Distribution of Phosphoinositides and Their Requirement for Auxin-Mediated Cell Polarity and Patterning in Arabidopsis

    NARCIS (Netherlands)

    Tejos, R.; Sauer, M.; Vanneste, S.; Palacios-Gomez, M.; Li, H.; Heilmann, M.; van Wijk, R.; Vermeer, J.E.M.; Heilmann, I.; Munnik, T.; Friml, J.

    2014-01-01

    Cell polarity manifested by asymmetric distribution of cargoes, such as receptors and transporters, within the plasma membrane (PM) is crucial for essential functions in multicellular organisms. In plants, cell polarity (re)establishment is intimately linked to patterning processes. Despite the

  1. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments.

    NARCIS (Netherlands)

    Gutierrez, R.; Lindeboom, J.J.; Paredez, A.R.; Emons, A.M.C.; Ehrhardt, D.W.

    2009-01-01

    Plant cell morphogenesis relies on the organization and function of two polymer arrays separated by the plasma membrane: the cortical microtubule cytoskeleton and cellulose microfibrils in the cell wall. Studies using in vivo markers confirmed that one function of the cortical microtubule array is

  2. Ectopic overexpression of a novel Glycine soja stress-induced plasma membrane intrinsic protein increases sensitivity to salt and dehydration in transgenic Arabidopsis thaliana plants.

    Science.gov (United States)

    Wang, Xi; Cai, Hua; Li, Yong; Zhu, Yanming; Ji, Wei; Bai, Xi; Zhu, Dan; Sun, Xiaoli

    2015-01-01

    Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5'-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants.

  3. 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.

  4. Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[OPEN

    Science.gov (United States)

    Vincent, Thomas R.; Avramova, Marieta; Canham, James; Higgins, Peter; Bilkey, Natasha; Mugford, Sam T.; Pitino, Marco; Toyota, Masatsugu

    2017-01-01

    A transient rise in cytosolic calcium ion concentration is one of the main signals used by plants in perception of their environment. The role of calcium in the detection of abiotic stress is well documented; however, its role during biotic interactions remains unclear. Here, we use a fluorescent calcium biosensor (GCaMP3) in combination with the green peach aphid (Myzus persicae) as a tool to study Arabidopsis thaliana calcium dynamics in vivo and in real time during a live biotic interaction. We demonstrate rapid and highly localized plant calcium elevations around the feeding sites of M. persicae, and by monitoring aphid feeding behavior electrophysiologically, we demonstrate that these elevations correlate with aphid probing of epidermal and mesophyll cells. Furthermore, we dissect the molecular mechanisms involved, showing that interplay between the plant defense coreceptor BRASSINOSTEROID INSENSITIVE-ASSOCIATED KINASE1 (BAK1), the plasma membrane ion channels GLUTAMATE RECEPTOR-LIKE 3.3 and 3.6 (GLR3.3 and GLR3.6), and the vacuolar ion channel TWO-PORE CHANNEL1 (TPC1) mediate these calcium elevations. Consequently, we identify a link between plant perception of biotic threats by BAK1, cellular calcium entry mediated by GLRs, and intracellular calcium release by TPC1 during a biologically relevant interaction. PMID:28559475

  5. Plasma membrane ATPases

    DEFF Research Database (Denmark)

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

    2011-01-01

    membrane include ABC transporters, vacuolar (V-type) H+ pumps, and P-type pumps. These pumps all utilize ATP as a fuel for energizing pumping. This review focuses on the physiological roles of plasma membrane P-type pumps, as they represent the major ATP hydrolytic activity in this membrane....

  6. COPT6 is a plasma membrane transporter that functions in copper homeostasis in Arabidopsis and is a novel target of SQUAMOSA promoter binding protein-like 7

    Science.gov (United States)

    Among the mechanisms controlling copper homeostasis in plants is the regulation of its uptake and tissue partitioning. Here we characterized a newly identified member of the conserved CTR/COPT family of copper transporters in Arabidopsis thaliana, COPT6. We showed that COPT6 resides at the plasma me...

  7. 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

    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...

  8. A bioassay-guided fractionation system to identify endogenous small molecules that activate plasma membrane H+-ATPase activity in Arabidopsis.

    Science.gov (United States)

    Han, Xiuli; Yang, Yongqing; Wu, Yujiao; Liu, Xiaohui; Lei, Xiaoguang; Guo, Yan

    2017-05-17

    Plasma membrane (PM) H+-ATPase is essential for plant growth and development. Various environmental stimuli regulate its activity, a process that involves many protein cofactors. However, whether endogenous small molecules play a role in this regulation remains unknown. Here, we describe a bio-guided isolation method to identify endogenous small molecules that regulate PM H+-ATPase activity. We obtained crude extracts from Arabidopsis seedlings with or without salt treatment and then purified them into fractions based on polarity and molecular mass by repeated column chromatography. By evaluating the effect of each fraction on PM H+-ATPase activity, we found that fractions containing the endogenous, free unsaturated fatty acids oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) extracted from salt-treated seedlings stimulate PM H+-ATPase activity. These results were further confirmed by the addition of exogenous C18:1, C18:2, or C18:3 in the activity assay. The ssi2 mutant, with reduced levels of C18:1, C18:2, and C18:3, displayed reduced PM H+-ATPase activity. Furthermore, C18:1, C18:2, and C18:3 directly bound to the C-terminus of the PM H+-ATPase AHA2. Collectively, our results demonstrate that the binding of free unsaturated fatty acids to the C-terminus of PM H+-ATPase is required for its activation under salt stress. The bio-guided isolation model described in this study could enable the identification of new endogenous small molecules that modulate essential protein functions, as well as signal transduction, in plants. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. The studies on the toxicity mechanism of environmentally hazardous natural (IAA) and synthetic (NAA) auxin--The experiments on model Arabidopsis thaliana and rat liver plasma membranes.

    Science.gov (United States)

    Hąc-Wydro, Katarzyna; Flasiński, Michał

    2015-06-01

    This paper concerns the studies towards membrane-damage effect of two auxins: indole-3-acetic acid - IAA and 1-naphthaleneacetic acid - NAA on plant (Arabidopsis thaliana) and animal (rat liver) model membranes. The foregoing auxins are plant growth regulators widely used in agriculture to control the quality of the crop. However, their accumulation in the environment makes them hazardous for the living organisms. The aim of our investigations was to compare the effect of natural (IAA) vs. synthetic (NAA) auxin on the organization of plant and animal model membranes and find a possible correlation between membrane-disturbing effect of these compounds and their toxicity. The collected data evidenced that auxins cause destabilization of membranes, decrease their condensation and weakens interactions of molecules. The alterations in the morphology of model systems were also noticed. The foregoing effects of auxins are concentration-dependent and additionally NAA was found to act on animal vs. plant membranes more selectively than IAA. Interestingly, both IAA and NAA induce the strongest disordering in model lipid system at the concentration, which is frequently reported as toxic to animal and plants. Based on the above findings it was proposed that membrane-damage effect induced by IAA and NAA may be important from the point of view of the mechanism of toxicity of these compounds and cannot be ignored in further investigations in this area. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. 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 ......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...

  11. Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

    DEFF Research Database (Denmark)

    Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta

    2008-01-01

    The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...

  12. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)

    ONOS

    2010-01-18

    Jan 18, 2010 ... Ewing and Bennet (1994) identi- fied at least 7 genes in tomato and Harper et al. (1994) identified 10 genes in Arabidopsis thaliana, indicating the presence of large families of H+-ATPase genes. In this report, we determine and localize the expression pattern of germination specific plasma membrane ...

  13. Metabolic labeling and membrane fractionation for comparative proteomic analysis of Arabidopsis thaliana suspension cell cultures.

    Science.gov (United States)

    Szymanski, Witold G; Kierszniowska, Sylwia; Schulze, Waltraud X

    2013-09-28

    Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% (1). Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient (2). Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K(15)NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest (3). By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or

  14. 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

  15. 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.

  16. Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

    Science.gov (United States)

    Astegno, Alessandra; Bonza, Maria Cristina; Vallone, Rosario; La Verde, Valentina; D'Onofrio, Mariapina; Luoni, Laura; Molesini, Barbara; Dominici, Paola

    2017-09-08

    Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca 2+ )-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca 2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca 2+ and Mg 2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca 2+ /Mg 2+ mixed binding sites and two low-affinity Ca 2+ -specific sites. Binding of Ca 2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca 2+ or Mg 2+ , stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca 2+ -ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca 2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca 2+ pumps. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. The "Arabidopsis cax3" mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H(+)-ATPase activity

    Science.gov (United States)

    Perturbing CAX1, an "Arabidopsis" vacuolar H(+)/Ca(2+) antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among "cax1" and "ca...

  18. Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+-associated opening of plasma membrane anion channels

    NARCIS (Netherlands)

    Jeworutzki, Elena; Roelfsema, M. Rob G.; Anschuetz, Uta; Krol, Elzbieta; Elzenga, J. Theo M.; Felix, Georg; Boller, Thomas; Hedrich, Rainer; Becker, Dirk

    P>The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe-associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN-SENSING 2 (FLS2) and EF-Tu receptor

  19. ARAMEMNON, a novel database for Arabidopsis integral membrane proteins

    DEFF Research Database (Denmark)

    Schwacke, Rainer; Schneider, Anja; van der Graaff, Eric

    2003-01-01

    A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs......, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM...

  20. 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.

  1. Calcium-dependent freezing tolerance in Arabidopsis involves membrane resealing via synaptotagmin SYT1.

    Science.gov (United States)

    Yamazaki, Tomokazu; Kawamura, Yukio; Minami, Anzu; Uemura, Matsuo

    2008-12-01

    Plant freezing tolerance involves the prevention of lethal freeze-induced damage to the plasma membrane. We hypothesized that plant freezing tolerance involves membrane resealing, which, in animal cells, is accomplished by calcium-dependent exocytosis following mechanical disruption of the plasma membrane. In Arabidopsis thaliana protoplasts, extracellular calcium enhanced not only freezing tolerance but also tolerance to electroporation, which typically punctures the plasma membrane. However, calcium did not enhance survival when protoplasts were exposed to osmotic stress that mimicked freeze-induced dehydration. Calcium-dependent freezing tolerance was also detected with leaf sections in which ice crystals intruded into tissues. Interestingly, calcium-dependent freezing tolerance was inhibited by extracellular addition of an antibody against the cytosolic region of SYT1, a homolog of synaptotagmin known to be a calcium sensor that initiates exocytosis. This inhibition indicates that the puncture allowing the antibody to flow into the cytoplasm occurs during freeze/thawing. Thus, we propose that calcium-dependent freezing tolerance results from resealing of the punctured site. Protoplasts or leaf sections isolated from Arabidopsis SYT1-RNA interference (RNAi) plants lost calcium-dependent freezing tolerance, and intact SYT1-RNAi plants had lower freezing tolerance than control plants. Taken together, these findings suggest that calcium-dependent freezing tolerance results from membrane resealing and that this mechanism involves SYT1 function.

  2. Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

    Science.gov (United States)

    Aoyama, Shoki; Terada, Saki; Sanagi, Miho; Hasegawa, Yoko; Lu, Yu; Morita, Yoshie; Chiba, Yukako; Sato, Takeo; Yamaguchi, Junji

    2017-09-09

    Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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.

  4. Arabidopsis

    Science.gov (United States)

    Khare, Deepa; Choi, Hyunju; Huh, Sung Un; Bassin, Barbara; Kim, Jeongsik; Martinoia, Enrico; Sohn, Kee Hoon; Paek, Kyung-Hee; Lee, Youngsook

    2017-07-11

    Plant pathogens cause huge yield losses. Plant defense often depends on toxic secondary metabolites that inhibit pathogen growth. Because most secondary metabolites are also toxic to the plant, specific transporters are needed to deliver them to the pathogens. To identify the transporters that function in plant defense, we screened Arabidopsis thaliana mutants of full-size ABCG transporters for hypersensitivity to sclareol, an antifungal compound. We found that atabcg34 mutants were hypersensitive to sclareol and to the necrotrophic fungi Alternaria brassicicola and Botrytis cinerea AtABCG34 expression was induced by A brassicicola inoculation as well as by methyl-jasmonate, a defense-related phytohormone, and AtABCG34 was polarly localized at the external face of the plasma membrane of epidermal cells of leaves and roots. atabcg34 mutants secreted less camalexin, a major phytoalexin in A thaliana , whereas plants overexpressing AtABCG34 secreted more camalexin to the leaf surface and were more resistant to the pathogen. When treated with exogenous camalexin, atabcg34 mutants exhibited hypersensitivity, whereas BY2 cells expressing AtABCG34 exhibited improved resistance. Analyses of natural Arabidopsis accessions revealed that AtABCG34 contributes to the disease resistance in naturally occurring genetic variants, albeit to a small extent. Together, our data suggest that AtABCG34 mediates camalexin secretion to the leaf surface and thereby prevents A brassicicola infection.

  5. Plasma membrane calcium ATPases are important components of receptor-mediated signaling in plant immune responses and development.

    NARCIS (Netherlands)

    Frei dit Frey, N.; Mbengue, M.; Kwaaitaal, M.A.C.J.; Nitsch, L.M.C.; Altenbach, D.; Häweker, H.; Lozano-Duran, R.; Njo, M.F.; Beeckman, T.; Huettel, B.; Borst, J.W.; Panstruga, R.; Robatzek, S.

    2012-01-01

    Plasma membrane-resident receptor kinases (RKs) initiate signaling pathways important for plant immunity and development. In Arabidopsis (Arabidopsis thaliana), the receptor for the elicitor-active peptide epitope of bacterial flagellin, flg22, is encoded by FLAGELLIN SENSING2 (FLS2), which promotes

  6. 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....

  7. Recycling from endosomes to the plasma membrane

    NARCIS (Netherlands)

    Dam, E.M. van

    2001-01-01

    Summary V Chapter?Summary Many membrane proteins are, after endocytic uptake, efficiently recycled back to the plasma membrane. The aim of the studies presented in this thesis was to determine pathways and molecular mechanisms that are involved in recycling. Plasma membrane-derived clathrin-coated

  8. 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

  9. Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

    Science.gov (United States)

    Kenderešová, Lucia; Staňová, Andrea; Pavlovkin, Ján; Ďurišová, Eva; Nadubinská, Miriam; Čiamporová, Milada; Ovečka, Miroslav

    2012-01-01

    Background and Aims Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa. Methods Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn2+. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (EM) of mature cortical root cells and changes in the pH of the liquid culture media were measured. Key Results Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn2+ concentrations than A. thaliana plants. Elevated Zn2+ concentrations in the culture medium induced rapid changes in EM. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn2+-induced lower pH. Conclusions The results indicate a similar mode of early reaction to Zn2+, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals. PMID:22645116

  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. 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...... 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...

  12. 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.

  13. 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.

  14. The plant plasma membrane H+-ATPase

    DEFF Research Database (Denmark)

    Ekberg, Kira

      The very high mobility of protons in aqueous solutions demands special features of membrane proton transporters to sustain efficient yet regulated proton transport across biological membranes. By the use of the chemical energy of ATP, plasma-membrane-embedded H+-ATPases extrude protons from cells...... 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...... to describe the basic molecular components that allow the plasma membrane proton H+-ATPase to carry out proton transport against large membrane potentials. Moreover, a completely new paradigm for post-translational activation of these proteins is presented. The talk will focus on the following themes...

  15. 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.

  16. Plasma membrane changes during programmed cell deaths.

    Science.gov (United States)

    Zhang, Yingying; Chen, Xin; Gueydan, Cyril; Han, Jiahuai

    2018-01-01

    Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers inflammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D (GSDMD) pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity.

  17. Ca(2+)-activated anion channels and membrane depolarizations induced by blue light and cold in Arabidopsis seedlings

    Science.gov (United States)

    Lewis, B. D.; Karlin-Neumann, G.; Davis, R. W.; Spalding, E. P.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The activation of an anion channel in the plasma membrane of Arabidopsis thaliana hypocotyls by blue light (BL) is believed to be a signal-transducing event leading to growth inhibition. Here we report that the open probability of this particular anion channel depends on cytoplasmic Ca2+ ([Ca2+]cyt) within the concentration range of 1 to 10 microM, raising the possibility that BL activates the anion channel by increasing [Ca2+]cyt. Arabidopsis seedlings cytoplasmically expressing aequorin were generated to test this possibility. Aequorin luminescence did not increase during or after BL, providing evidence that Ca2+ does not play a second-messenger role in the activation of anion channels. However, cold shock simultaneously triggered a large increase in [Ca2+]cyt and a 110-mV transient depolarization of the plasma membrane. A blocker of the anion channel, 5-nitro-2-(3-phenylpropylamino)-benzoic acid, blocked 61% of the cold-induced depolarization without affecting the increase in [Ca2+]cyt. These data led us to propose that cold shock opens Ca2+ channels at the plasma membrane, allowing an inward, depolarizing Ca2+ current. The resulting large increase in [Ca2+]cyt activates the anion channel, which further depolarizes the membrane. Although an increase in [Ca2+]cyt may activate anion channels in response to cold, it appears that BL does so via a Ca(2+)-independent pathway.

  18. 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.

  19. 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.

  20. Dynamics of membrane potential variation and gene expression induced by Spodoptera littoralis, Myzus persicae, and Pseudomonas syringae in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Irene Bricchi

    Full Text Available BACKGROUND: Biotic stress induced by various herbivores and pathogens invokes plant responses involving different defense mechanisms. However, we do not know whether different biotic stresses share a common response or which signaling pathways are involved in responses to different biotic stresses. We investigated the common and specific responses of Arabidopsis thaliana to three biotic stress agents: Spodoptera littoralis, Myzus persicae, and the pathogen Pseudomonas syringae. METHODOLOGY/PRINCIPAL FINDINGS: We used electrophysiology to determine the plasma membrane potential (V(m and we performed a gene microarray transcriptome analysis on Arabidopsis upon either herbivory or bacterial infection. V(m depolarization was induced by insect attack; however, the response was much more rapid to S. littoralis (30 min -2 h than to M. persicae (4-6 h. M. persicae differentially regulated almost 10-fold more genes than by S. littoralis with an opposite regulation. M. persicae modulated genes involved in flavonoid, fatty acid, hormone, drug transport and chitin metabolism. S. littoralis regulated responses to heat, transcription and ion transport. The latest Vm depolarization (16 h was found for P. syringae. The pathogen regulated responses to salicylate, jasmonate and to microorganisms. Despite this late response, the number of genes differentially regulated by P. syringae was closer to those regulated by S. littoralis than by M. persicae. CONCLUSIONS/SIGNIFICANCE: Arabidopsis plasma membranes respond with a V(m depolarization at times depending on the nature of biotic attack which allow setting a time point for comparative genome-wide analysis. A clear relationship between V(m depolarization and gene expression was found. At V(m depolarization timing, M. persicae regulates a wider array of Arabidopsis genes with a clear and distinct regulation than S. littoralis. An almost completely opposite regulation was observed between the aphid and the pathogen

  1. Dynamics of Membrane Potential Variation and Gene Expression Induced by Spodoptera littoralis, Myzus persicae, and Pseudomonas syringae in Arabidopsis

    Science.gov (United States)

    Bricchi, Irene; Bertea, Cinzia M.; Occhipinti, Andrea; Paponov, Ivan A.; Maffei, Massimo E.

    2012-01-01

    Background Biotic stress induced by various herbivores and pathogens invokes plant responses involving different defense mechanisms. However, we do not know whether different biotic stresses share a common response or which signaling pathways are involved in responses to different biotic stresses. We investigated the common and specific responses of Arabidopsis thaliana to three biotic stress agents: Spodoptera littoralis, Myzus persicae, and the pathogen Pseudomonas syringae. Methodology/Principal Findings We used electrophysiology to determine the plasma membrane potential (Vm) and we performed a gene microarray transcriptome analysis on Arabidopsis upon either herbivory or bacterial infection. Vm depolarization was induced by insect attack; however, the response was much more rapid to S. littoralis (30 min −2 h) than to M. persicae (4–6 h). M. persicae differentially regulated almost 10-fold more genes than by S. littoralis with an opposite regulation. M. persicae modulated genes involved in flavonoid, fatty acid, hormone, drug transport and chitin metabolism. S. littoralis regulated responses to heat, transcription and ion transport. The latest Vm depolarization (16 h) was found for P. syringae. The pathogen regulated responses to salicylate, jasmonate and to microorganisms. Despite this late response, the number of genes differentially regulated by P. syringae was closer to those regulated by S. littoralis than by M. persicae. Conclusions/Significance Arabidopsis plasma membranes respond with a Vm depolarization at times depending on the nature of biotic attack which allow setting a time point for comparative genome-wide analysis. A clear relationship between Vm depolarization and gene expression was found. At Vm depolarization timing, M. persicae regulates a wider array of Arabidopsis genes with a clear and distinct regulation than S. littoralis. An almost completely opposite regulation was observed between the aphid and the pathogen, with the former

  2. 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.

  3. 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.

  4. Relationship between sperm plasma membrane integrity and ...

    African Journals Online (AJOL)

    Sperm quality plays an important role in determining fertility. The aim of the study was to examine the relationship between sperm plasma membrane integrity and morphology, and fertility following artificial insemination (AI). A total of 16 ejaculates were collected from three Large White boars using the gloved hand ...

  5. 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.

  6. Investigating membrane-bound Argonaute functions in Arabidopsis

    DEFF Research Database (Denmark)

    Barghetti, Andrea

    by direct cleavage or by inhibition of their translation. Arabidopsis genome encode for 10 different AGO proteins, of which AGO1 is the main effector of post-transcriptional gene silencing mediated by sRNA. Importantly, a fraction of AGO1 population is associated with endomembranes, in particular...

  7. Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

    Directory of Open Access Journals (Sweden)

    Kasaras Alexis

    2012-04-01

    Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

  8. Mechanotransduction through the plasma membrane & cytoskeleton

    Science.gov (United States)

    Haase, Kristina; Pelling, Andrew

    2012-02-01

    Mechanical forces initiate immediate and long-term changes in cells; however the exact mechanisms remain unclear, albeit crucial for understanding the pathology of disease. We used combined confocal and atomic force microscopy (AFM) to investigate changes in cell morphology and elasticity in response to a mechanical stimulus. The AFM was used as a nano-indentor to gauge the response of the membrane and cytoskeleton (CSK) of HeLa cells. We observed their viscoelastic nature by probing cells transfected with a green fluorescent protein localized at the plasma membrane. Inhibition of acto-myosin contractility (AMc) resulted in a significant decrease of cellular elasticity, and a corresponding increase in mean deformation. We also investigated the rate at which the membrane and CSK deform and relax in response to a local force. The response to a local perturbation is nearly instantaneous for control cells and shows no statistical difference when compared to cells treated with CSK-inhibiting drugs. Inhibition of AMc affects the rate of recovery, in comparison to control cells which recover quite quickly (30-60s). Overall, we demonstrated short and long-term deformation and subsequent recovery of both the cell membrane and actin network in response to a local force.

  9. 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.

  10. Hydrogen Production from Ammonia Using a Plasma Membrane Reactor

    Directory of Open Access Journals (Sweden)

    Shinji Kambara

    2016-06-01

    Full Text Available In this study, an efficient method for using pulsed plasma to produce hydrogen from ammonia was developed. An original pulsed plasma reactor with a hydrogen separation membrane was developed for efficient hydrogen production, and its hydrogen production performance was investigated. Hydrogen production in the plasma was affected by the applied voltage and flow rate of ammonia gas. The maximum hydrogen production flow rate of a typical plasma reactor was 8.7 L/h, whereas that of the plasma membrane reactor was 21.0 L/h. We found that ammonia recombination reactions in the plasma controlled hydrogen production in the plasma reactor. In the plasma membrane reactor, a significant increase in hydrogen production was obtained because ammonia recombination reactions were inhibited by the permeation of hydrogen radicals generated in the plasma through a palladium alloy membrane. The energy efficiency was 4.42 mol-H2/kWh depending on the discharge power.

  11. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization

    DEFF Research Database (Denmark)

    Schiøtt, Morten; Romanowsky, Shawn M; Bækgaard, Lone

    2004-01-01

    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome...... encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor......-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential...

  12. 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.

  13. Selective photosensitizer delivery into plasma membrane for effective photodynamic therapy.

    Science.gov (United States)

    Kim, Jiyoung; Santos, Olavo Amorim; Park, Ji-Ho

    2014-10-10

    Subcellular localization of photosensitizers (PSs) determines the therapeutic efficacy in the photodynamic therapy. However, among the subcellular compartments, there has been little effort to deliver the PSs selectively into the plasma membrane and examine the phototherapeutic efficacy of membrane-localized PSs. Here, we developed a liposomal delivery system to localize the hydrophobic PSs selectively into the plasma membrane. The membrane fusogenic liposomes (MFLs), the membrane of which is engineered to fuse with the plasma membrane, was prepared for the membrane localization of PSs. The phototherapeutic efficacy of cells treated with ZnPc-loaded MFLs was superior over that of cells treated with ZnPc-loaded non-fusogenic liposomes, which is the conventional liposomal formulation that delivers the PSs into the intracellular compartments via endocytosis. The membrane localization of ZnPc molecules led to rapid membrane disruption upon irradiation and subsequent necrosis-like cell death. The membrane-localized generation of reactive oxygen species in the cells treated with ZnPc-loaded MFLs was likely to account for the effective disruption of plasma membrane. Thus, this work provides a novel delivery method to localize the PSs selectively into the plasma membrane with the enhanced phototherapeutic efficacy. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Vesicular and Plasma Membrane Transporters for Neurotransmitters

    Science.gov (United States)

    Blakely, Randy D.; Edwards, Robert H.

    2012-01-01

    The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity. PMID:22199021

  15. Plasma membranes from insect midgut cells

    Directory of Open Access Journals (Sweden)

    Walter R. Terra

    2006-06-01

    Full Text Available Plasma membranes from insect midgut cells are separated into apical and basolateral domains. The apical domain is usually modified into microvilli with a molecular structure similar to other animals. Nevertheless, the microvillar structure should differ in some insects to permit the traffic inside them of secretory vesicles that may budd laterally or pinch-off from the tips of microvilli. Other microvillar modifications are associated with proton-pumping or with the interplay with an ensheathing lipid membrane (the perimicrovilllar membrane observed in the midgut cells of hemipterans (aphids and bugs. The perimicrovillar membranes are thought to be involved in amino acid absorption from diluted diets. The microvillar and perimicrovillar membranes have densities (and protein content that depend on the insect taxon. The role played by the microvillar and perimicrovillar proteins in insect midgut physiology is reviewed here trying to provide a coherent picture of data and highlighting further research areas.As membranas plasmáticas das células intestinais dos insetos apresentam um domínio apical e outro basal. O domínio apical é geralmente modificado em microvilosidades com organização molecular similar a de outros animais, embora possam diferir naqueles insetos que apresentam vesículas secretoras em trânsito que brotam lateralmente ou destacam-se das extremidades das microvilosidades. Outras modificações microvilares estão associadas a bombeamento de prótons ou a interrelações com uma membrana lipídica (a membrana perimicrovilar que reveste as microvilosidades de células intestinais de hemípteros (pulgões e percevejos. Admite-se que as membranas perimicrovilares estejam envolvidas na absorção de aminoácidos a partir de dietas diluídas. As membranas microvilares e perimicrovilares tem densidades distintas (e conteúdo protéico que dependem do táxon do inseto. O papel desempenhado pelas proteínas microvilares e

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

    African Journals Online (AJOL)

    The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RTPCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat embryos.

  17. A plasma membrane H ATPase gene is germination- induced in ...

    African Journals Online (AJOL)

    ONOS

    2010-01-18

    Jan 18, 2010 ... The expression pattern of a germination specific plasma membrane H+-ATPase was analyzed by RT-. PCR and in situ RNA hybridization methods. RT-PCR results revealed that germination specific plasma membrane H+-ATPase accumulation was detectable in all organs and tissues of germinating wheat.

  18. Channelopathies linked to plasma membrane phosphoinositides

    Science.gov (United States)

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul

    2014-01-01

    The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

  19. Large Deformation Mechanics of Plasma Membrane Chained Vesicles in Cells

    Science.gov (United States)

    Kosawada, Tadashi; Sanada, Kouichi; Takano, Tetsuo

    The clathrin-coated pits, vesicles and chained vesicles on the inner surface of the plasma membrane facilitate the cell to transport specific extracellular macromolecules. This cellular process is strongly involved with large mechanical deformations of the plasma membrane accompanied by changes in membrane curvature. The assembly of the clathrin coat is thought to provide curvature into the membrane. Hence, effects of in-plane shear elasticity due to these coat structure may be significant on the vesicular mechanics. In this study, large deformation mechanics of plasma membrane chained vesicles in cells have been formulated based on minimization of bending and in-plane shear strain energy of the membrane. Effects of outer surrounding cytoplasmic flat membrane upon mechanically stable shapes of the vesicles were revealed, while effects of in-plane shear elasticity were partly discussed.

  20. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  1. A membrane protein / signaling protein interaction network for Arabidopsis version AMPv2

    Directory of Open Access Journals (Sweden)

    Sylvie Lalonde

    2010-09-01

    Full Text Available Interactions between membrane proteins and the soluble fraction are essential for signal transduction and for regulating nutrient transport. To gain insights into the membrane-based interactome, 3,852 open reading frames (ORFs out of a target list of 8,383 representing membrane and signaling proteins from Arabidopsis thaliana were cloned into a Gateway compatible vector. The mating-based split-ubiquitin system was used to screen for potential protein-protein interactions (pPPIs among 490 Arabidopsis ORFs. A binary robotic screen between 142 receptor-like kinases, 72 transporters, 57 soluble protein kinases and phosphatases, 40 glycosyltransferases, 95 proteins of various functions and 89 proteins with unknown function detected 387 out of 90,370 possible PPIs. A secondary screen confirmed 343 (of 387 pPPIs between 179 proteins, yielding a scale-free network (r2=0.863. Eighty of 142 transmembrane receptor-like kinases (RLK tested positive, identifying three homomers, 63 heteromers and 80 pPPIs with other proteins. Thirty-one out of 142 RLK interactors (including RLKs had previously been found to be phosphorylated; thus interactors may be substrates for respective RLKs. None of the pPPIs described here had been reported in the major interactome databases, including potential interactors of G protein-coupled receptors, phospholipase C, and AMT ammonium transporters. Two RLKs found as putative interactors of AMT1;1 were independently confirmed using a split luciferase assay in Arabidopsis protoplasts. These RLKs may be involved in ammonium-dependent phosphorylation of the C-terminus and regulation of ammonium uptake activity. The robotic screening method established here will enable a systematic analysis of membrane protein interactions in fungi, plants and metazoa.

  2. 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.

  3. 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 amphiphilic...... 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....

  4. Macroscopic domain formation in the platelet plasma membrane

    DEFF Research Database (Denmark)

    Bali, Rachna; Savino, Laura; Ramirez, Diego A.

    2009-01-01

    phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents......There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large...

  5. 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

  6. Isolation of Arabidopsis thylakoid membranes and their use for in vitro protein insertion or transport assays.

    Science.gov (United States)

    Bals, Thomas; Schünemann, Danja

    2011-01-01

    This chapter focuses on the techniques of chloroplast isolation; their fractionation into envelopes, stroma, and thylakoids; and their further use for in vitro protein transport assays. In addition to the isolation of thylakoids, this chapter also describes the experimental steps of both protein translocation across the thylakoid membrane and protein integration into the membrane. Protein translocation and integration can be analysed by the radioactive labelling of substrate proteins using an in vitro transcription and translation system. The translocated or integrated proteins can then be detected by autoradiography. Our protocol allows the analysis of these transport systems in wild-type Arabidopsis or mutants that lack or overexpress soluble or membrane transport factors that could be of potential interest.

  7. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization.

    Science.gov (United States)

    Schiøtt, Morten; Romanowsky, Shawn M; Baekgaard, Lone; Jakobsen, Mia Kyed; Palmgren, Michael G; Harper, Jeffrey F

    2004-06-22

    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants.

  8. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization...

  9. 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.

  10. 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

  11. 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...

  12. Nuclear membrane localization during pollen development and apex-focused polarity establishment of SYP124/125 during pollen germination in Arabidopsis thaliana.

    Science.gov (United States)

    Ichikawa, Mie; Iwano, Megumi; Sato, Masa H

    2015-12-01

    Establishment of apex-polarity. Elongation of the pollen tube is a highly coordinated process involving polarized secretion of cell wall and membrane materials to the apical region. We investigated changes in the localization of soluble NSF attachment proteins (SNAREs) in developing pollen grains and the pollen tube for transgenic Arabidopsis expressing pollen-specific plasma-membrane Qa-SNAREs (SYP124, 125 and 131) fused with the green fluorescent protein (GFP). The expression of SYP124 and SYP125 was firstly detected in the microspore nuclear membrane during pollen mitosis II. Although SYP124, 125 and 131 accumulated throughout the cytosol in the mature pollen grain, GFP-SYP124 and GFP-SYP125 were highly concentrated in the apical or subapical regions of the elongating pollen tube with slightly different localization patterns, whereas GFP-SYP131 was uniformly localized to the plasma membrane of the pollen tube. The apex-focused polarity of GFP-SYP125 was established coincident with formation of a Ca(2+) gradient before pollen germination. These results suggest that SNAREs function differentially in the same cells and that at least two distinct membrane transport pathways are involved in the pollen development and the pollen tube germination and elongation.

  13. 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.

  14. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    Directory of Open Access Journals (Sweden)

    Qingqing Lin

    Full Text Available Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM and/or phosphatidylcholine (PC outside/phosphatidylethanolamine (PE and phosphatidylserine (PS inside, and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm" vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  15. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry.

    Science.gov (United States)

    Lin, Qingqing; London, Erwin

    2014-01-01

    Lipid asymmetry, the difference in lipid distribution across the lipid bilayer, is one of the most important features of eukaryotic cellular membranes. However, commonly used model membrane vesicles cannot provide control of lipid distribution between inner and outer leaflets. We recently developed methods to prepare asymmetric model membrane vesicles, but facile incorporation of a highly controlled level of cholesterol was not possible. In this study, using hydroxypropyl-α-cyclodextrin based lipid exchange, a simple method was devised to prepare large unilamellar model membrane vesicles that closely resemble mammalian plasma membranes in terms of their lipid composition and asymmetry (sphingomyelin (SM) and/or phosphatidylcholine (PC) outside/phosphatidylethanolamine (PE) and phosphatidylserine (PS) inside), and in which cholesterol content can be readily varied between 0 and 50 mol%. We call these model membranes "artificial plasma membrane mimicking" ("PMm") vesicles. Asymmetry was confirmed by both chemical labeling and measurement of the amount of externally-exposed anionic lipid. These vesicles should be superior and more realistic model membranes for studies of lipid-lipid and lipid-protein interaction in a lipid environment that resembles that of mammalian plasma membranes.

  16. Dynamic Mechanical Responses of Arabidopsis Thylakoid Membranes during PSII-Specific Illumination

    Science.gov (United States)

    Clausen, Casper H.; Brooks, Matthew D.; Li, Tai-De; Grob, Patricia; Kemalyan, Gigi; Nogales, Eva; Niyogi, Krishna K.; Fletcher, Daniel A.

    2014-01-01

    Remodeling of thylakoid membranes in response to illumination is an important process for the regulation of photosynthesis. We investigated the thylakoid network from Arabidopsis thaliana using atomic force microscopy to capture dynamic changes in height, elasticity, and viscosity of isolated thylakoid membranes caused by changes in illumination. We also correlated the mechanical response of the thylakoid network with membrane ultrastructure using electron microscopy. We find that the elasticity of the thylakoid membranes increases immediately upon PSII-specific illumination, followed by a delayed height change. Direct visualization by electron microscopy confirms that there is a significant change in the packing repeat distance of the membrane stacks in response to illumination. Although experiments with Gramicidin show that the change in elasticity depends primarily on the transmembrane pH gradient, the height change requires both the pH gradient and STN7-kinase-dependent phosphorylation of LHCII. Our studies indicate that lumen expansion in response to illumination is not simply a result of the influx of water, and we propose a dynamic model in which protein interactions within the lumen drive these changes. PMID:24806918

  17. 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

  18. Enquiry into the Topology of Plasma Membrane-Localized PIN Auxin Transport Components.

    Science.gov (United States)

    Nodzyński, Tomasz; Vanneste, Steffen; Zwiewka, Marta; Pernisová, Markéta; Hejátko, Jan; Friml, Jiří

    2016-11-07

    Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Miscibility Critical Points in Plasma Membranes

    Science.gov (United States)

    Machta, Benjamin; Veatch, Sarah; Papanikolaou, Stefanos; Sethna, James P.

    2009-03-01

    Lipid bilayers surround all cells and are home to a host of proteins and lipids that mediate interactions between the cell and its environment. Recent experimental work has shown that simple membranes composed of three lipid components show complex phase behavior at temperatures in the physiological range. For example, two liquid phases and a gel or solid phase are seen, and a second order phase transition with Ising critical behavior can be reached at a boundary of the liquid-liquid coexistence region [1]. Surprisingly, membrane vesicles isolated from living cells can be tuned with a single parameter (temperature) to criticality [1]. This suggests that cell membranes in vivo sit near miscibility critical points, and may help explain some of the paradoxes associated with putative lipid rafts proposed in other experiments. Here we report on work mapping phase diagrams for the simple membranes utilizing NMR and microscopy data. In addition, we use canonical models of phase transitions to understand the qualitative features of the membranes. Finally we explore ideas from information theory and self organized criticality to understand how and why real cells maintain a membrane near criticality. [1] Honerkamp-Smith, Veatch, and Keller, Biochim Biophys Acta. 2008 (in press)

  20. Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

    Science.gov (United States)

    Laganowsky, Arthur; Gómez, Stephen M; Whitelegge, Julian P; Nishio, John N

    2009-04-13

    The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

  1. Rapid regulation of the plasma membrane H⁺-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa.

    Science.gov (United States)

    Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey

    2015-02-01

    The activity of H(+)-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na(+) exclusion via Na(+)/H(+) exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H(+)-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. The kinetics of salt-induced net H(+), Na(+) and K(+) fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (-144 ± 3·3, -138 ± 5·4 and -128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H(+) efflux, Na(+) efflux and K(+) retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H(+) efflux was most pronounced in the root elongation zone. In contrast, H(+)-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant's ability to rapidly upregulate plasma membrane H(+)-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to

  2. Rapid regulation of the plasma membrane H+-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa

    Science.gov (United States)

    Bose, Jayakumar; Rodrigo-Moreno, Ana; Lai, Diwen; Xie, Yanjie; Shen, Wenbiao; Shabala, Sergey

    2015-01-01

    Background and Aims The activity of H+-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na+ exclusion via Na+/H+ exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H+-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. Methods The kinetics of salt-induced net H+, Na+ and K+ fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. Key Results Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (−144 ± 3·3, −138 ± 5·4 and −128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H+ efflux, Na+ efflux and K+ retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H+ efflux was most pronounced in the root elongation zone. In contrast, H+-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. Conclusions Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant’s ability to rapidly upregulate plasma membrane H+-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative

  3. The evolutionarily conserved protein PHOTOSYNTHESIS AFFECTED MUTANT71 is required for efficient manganese uptake at the thylakoid membrane in Arabidopsis

    DEFF Research Database (Denmark)

    Schneider, Anja; Steinberger, Iris; Herdean, Andrei

    2016-01-01

    lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn2+ and Ca2+ homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced...

  4. 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.

  5. 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.

  6. NADH-Ferricyanide Reductase of Leaf Plasma Membranes 1

    Science.gov (United States)

    Askerlund, Per; Laurent, Pascal; Nakagawa, Hiroki; Kader, Jean-Claude

    1991-01-01

    Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber

  7. Plasma membrane proteome analysis identifies a role of barley Membrane Steroid Binding Protein in root architecture response to salinity.

    Science.gov (United States)

    Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarrayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

    2018-01-31

    Although physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane (PM) proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 PM proteins were identified by mass spectrometry, of which 182 were either cultivar- or salinity stress-responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol-binding (a GTPase-activating protein for the ADP ribosylation factor, ZIGA2, and a membrane steroid binding protein, MSBP) or in phospholipid synthesis (phosphoethanolamine methyltransferase, PEAMT). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, while knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity and root tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. This article is protected by copyright. All rights reserved.

  8. Detection of glycoproteins in the Acanthamoeba plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Paatero, G.I.L. (Abo Akademi (Finland)); Gahmberg, C.G. (Univ. of Helsinki (Finland))

    1988-11-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by {sup 125}I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB{sup 3}H{sub 4} and galactose oxidase/NaB{sup 3}H{sub 4} labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M{sub r} of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with ({sup 35}S)methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis.

  9. Detection of glycoproteins in the Acanthamoeba plasma membrane

    International Nuclear Information System (INIS)

    Paatero, G.I.L.; Gahmberg, C.G.

    1988-01-01

    In the present study the authors have shown that glycoproteins are present in the plasma membrane of Acanthamoeba castellanii by utilizing different radioactive labeling techniques. Plasma membrane proteins in the amoeba were iodinated by 125 I-lactoperoxidase labeling and the solubilized radiolabeled glycoproteins were separated by lectin-Sepharose affinity chromatography followed by polyacrylamide gel electrophoresis. The periodate/NaB 3 H 4 and galactose oxidase/NaB 3 H 4 labeling techniques were used for labeling of surface carbohydrates in the amoeba. Several surface-labeled glycoproteins were observed in addition to a diffusely labeled region with M r of 55,000-75,000 seen on electrophoresis, which could represent glycolipids. The presence of glycoproteins in the plasma membrane of Acanthamoeba castellanii was confirmed by metabolic labeling with [ 35 S]methionine followed by lectin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis

  10. 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...... other P-type ATPase. Phosphosites were almost exclusively (9 of 10) in the terminal regulatory domains of the pumps. The AHA2 isoform was subsequently expressed in the yeast Saccharomyces cerevisiae. The plant protein was phosphorylated at multiple sites in yeast, and surprisingly, seven of nine...... 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...

  11. 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.

  12. Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

    Science.gov (United States)

    Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

    2015-03-01

    In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

  13. 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

    2015-01-21

    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.

  14. 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

  15. Phenylarsine Oxide Inhibits the Fusicoccin-Induced Activation of Plasma Membrane H+-ATPase1

    Science.gov (United States)

    Olivari, Claudio; Albumi, Cristina; Pugliarello, Maria Chiara; De Michelis, Maria Ida

    2000-01-01

    To investigate the mechanism by which fusicoccin (FC) induces the activation of the plasma membrane (PM) H+-ATPase, we used phenylarsine oxide (PAO), a known inhibitor of protein tyrosine-phosphatases. PAO was supplied in vivo in the absence or presence of FC to radish (Raphanus sativus L.) seedlings and cultured Arabidopsis cells prior to PM extraction. Treatment with PAO alone caused a slight decrease of PM H+-ATPase activity and, in radish, a decrease of PM-associated 14-3-3 proteins. When supplied prior to FC, PAO drastically inhibited FC-induced activation of PM H+-ATPase, FC binding to the PM, and the FC-induced increase of the amount of 14-3-3 associated with the PM. On the contrary, PAO was completely ineffective on all of the above-mentioned parameters when supplied after FC. The H+-ATPase isolated from PAO-treated Arabidopsis cells maintained the ability to respond to FC if supplied with exogenous, nonphosphorylated 14-3-3 proteins. Altogether, these results are consistent with a model in which the dephosphorylated state of tyrosine residues of a protein(s), such as 14-3-3 protein, is required to permit FC-induced association between the 14-3-3 protein and the PM H+-ATPase. PMID:10677439

  16. 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...

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

    Czech Academy of Sciences Publication Activity Database

    Opekarová, Miroslava; Malínský, Jan; Tanner, W.

    2010-01-01

    Roč. 12, č. 1 (2010), s. 94-98 ISSN 1435-8603 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50390512 Keywords : Confocal microscopy * plasma membrane * raft-like micro domains Subject RIV: EE - Microbiology, Virology Impact factor: 2.409, year: 2010

  18. 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

  19. 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

  20. A plasma membrane association module in yeast amino acid transporters

    NARCIS (Netherlands)

    Popov-Čeleketić, Dušan; Bianchi, Frans; Ruiz, Stephanie J; Meutiawati, Febrina; Poolman, Bert

    2016-01-01

    Amino acid permeases (AAPs) in the plasma membrane (PM) of Saccharomyces cerevisiae are responsible for the uptake of amino acids and involved in regulation of their cellular levels. Here, we report on a strong and complex module for PM association found in the C-terminal tail of AAPs. Using in

  1. 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

  2. Calcium pumps of plasma membrane and cell interior

    DEFF Research Database (Denmark)

    Strehler, Emanuel E; Treiman, Marek

    2004-01-01

    /endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue...

  3. 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.

  4. Plasma membrane aquaporins mediates vesicle stability in broccoli.

    Science.gov (United States)

    Martínez-Ballesta, Maria Del Carmen; García-Gomez, Pablo; Yepes-Molina, Lucía; Guarnizo, Angel L; Teruel, José A; Carvajal, Micaela

    2018-01-01

    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.

  5. 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.

    2017-06-01

    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.

  6. 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)

  7. 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.

  8. Measurement of autoantibodies against human eye muscle plasma membranes in Graves' ophthalmopathy.

    OpenAIRE

    Faryna, M; Nauman, J; Gardas, A

    1985-01-01

    Antibodies that reacted with plasma membranes of human eye muscle but showed no binding to plasma membranes of human skeletal muscle were identified in serum of patients with Graves' ophthalmopathy. Rabbit antithyroglobulin serum at a dilution of 1 X 10(-3) to 20 X 10(-3) had no effect on the binding of these antibodies to eye muscle membrane antigens. There was no correlation between antihuman eye muscle plasma membrane antibodies and antihuman thyroid membrane antibodies or antibodies again...

  9. 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

  10. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

    Science.gov (United States)

    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  11. Manufacturing of metal supported BSCF membranes by spark plasma sintering

    OpenAIRE

    Laptev, Alexander; Bram, Martin; Zivcec, Maria; Baumann, Stefan; Jarligo, Maria Ophelia; Sebold, Doris; Pfaff, Ewald; Broeckmann, Christoph

    2013-01-01

    Spark plasma sintering (SPS), also known as field assisted sintering technique (FAST), is a relatively new method for rapid consolidation of metallic or ceramic powders. In the present work, its suitability for the manufacturing of metal supported Ba0.5Sr0.5Co0.8Fe0.2O3-δ (BSCF) based membrane by co-sintering of functional ceramic BSCF layer and porous metallic support has been investigated. The BSCF based membranes are highly attractive for oxygen separation from air due to mixed ionic and e...

  12. 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.

  13. 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).

  14. 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.

  15. AtCHX13 is a plasma membrane K(+) transporter

    Science.gov (United States)

    Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K(+) required for growth and development is poorly understood. The Arabidopsis ("Arabidopsis thaliana") genome contains numerous cation:proton antiporte...

  16. AtCHX13 is a plasma membrane K+ transporter

    Science.gov (United States)

    Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (...

  17. 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.

  18. 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.

  19. 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

  20. Regulation of the plasma membrane proton pump (H+-ATPase) by phosphorylation

    Science.gov (United States)

    Haruta, Miyoshi; Gray, William M.; Sussman, Michael R.

    2015-01-01

    In plants and fungi, energetics at the plasma membrane is provided by a large protonmotive force (PMF) generated by the family of P-type ATPases specialized for proton transport (commonly called PM H+-ATPases or, in Arabidopsis, AHAs for Arabidopsis H+-ATPases). Studies have demonstrated that this 100-kDa protein is essential for plant growth and development. Posttranslational modifications of the H+-ATPase play critical roles in its regulation. Phosphorylation of several Thr and Ser residues within the carboxy terminal regulatory domain composed of ~ 100 amino acids change in response to environmental stimuli, endogenous hormones, and nutrient conditions. Recently developed mass spectrometric technologies provide a means to carefully quantify these changes in H+-ATPase phosphorylation at the different sites. These chemical modifications can then be genetically tested in planta by complementing the loss-of-function aha mutants with phosphomimetic mutations. Interestingly, recent data suggest that phosphatase-mediated changes in PM H+-ATPase phosphorylation are important in mediating auxin-regulated growth. Thus, as with another hormone (abscisic acid), dephosphorylation by phosphatases, rather than kinase mediated phosphorylation, may be an important focal point for regulation during plant signal transduction. Although interactions with other proteins have also been implicated in ATPase regulation, the very hydrophobic nature and high concentration of this polytopic protein presents special challenges in evaluating the biological significance of these interactions. Only by combining biochemical and genetic experiments can we attempt to meet these challenges to understand the essential molecular details by which this protein functions in planta. PMID:26476298

  1. Regulation of the plasma membrane proton pump (H(+)-ATPase) by phosphorylation.

    Science.gov (United States)

    Haruta, Miyoshi; Gray, William M; Sussman, Michael R

    2015-12-01

    In plants and fungi, energetics at the plasma membrane is provided by a large protonmotive force (PMF) generated by the family of P-type ATPases specialized for proton transport (commonly called PM H(+)-ATPases or, in Arabidopsis, AHAs for Arabidopsis H(+)-ATPases). Studies have demonstrated that this 100-kDa protein is essential for plant growth and development. Posttranslational modifications of the H(+)-ATPase play crucial roles in its regulation. Phosphorylation of several Thr and Ser residues within the carboxy terminal regulatory domain composed of ∼100 amino acids change in response to environmental stimuli, endogenous hormones, and nutrient conditions. Recently developed mass spectrometric technologies provide a means to carefully quantify these changes in H(+)-ATPase phosphorylation at the different sites. These chemical modifications can then be genetically tested in planta by complementing the loss-of-function aha mutants with phosphomimetic mutations. Interestingly, recent data suggest that phosphatase-mediated changes in PM H(+)-ATPase phosphorylation are important in mediating auxin-regulated growth. Thus, as with another hormone (abscisic acid), dephosphorylation by phosphatases, rather than kinase mediated phosphorylation, may be an important focal point for regulation during plant signal transduction. Although interactions with other proteins have also been implicated in ATPase regulation, the very hydrophobic nature and high concentration of this polytopic protein presents special challenges in evaluating the biological significance of these interactions. Only by combining biochemical and genetic experiments can we attempt to meet these challenges to understand the essential molecular details by which this protein functions in planta. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Artificial plasma membrane models based on lipidomic profiling.

    Science.gov (United States)

    Essaid, Donia; Rosilio, Véronique; Daghildjian, Katia; Solgadi, Audrey; Vergnaud, Juliette; Kasselouri, Athena; Chaminade, Pierre

    2016-11-01

    Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. 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

    -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...... the functional unit of ATP-coupled proton transport across the plasma membrane, and the structure is locked in a functional state not previously observed in P-type ATPases. The transmembrane domain reveals a large cavity, which is likely to be filled with water, located near the middle of the membrane plane...

  4. The basidiomycete Ustilago maydis has two plasma membrane H⁺-ATPases related to fungi and plants.

    Science.gov (United States)

    Robles-Martínez, Leobarda; Pardo, Juan Pablo; Miranda, Manuel; Mendez, Tavis L; Matus-Ortega, Macario Genaro; Mendoza-Hernández, Guillermo; Guerra-Sánchez, Guadalupe

    2013-10-01

    The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

  5. 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.

  6. 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...... a huge amount of energy in form of ATP, to pump out protons. To avoid complete energy depletion in the cells, tight regulation of the PM H+-ATPase is a necessity. The proteins two terminal domains have been identified as autoinhibitory domains that regulate the pumping activity, but due to lack of a high...... in mammalian cells and it has been speculated if they have a similar function in plants. In this thesis we show, that plant PM H+-ATPases are receptors for lysophospholipids and the autoinhibitory terminal inhibition is released upon lysophospholipid binding. Finally, we have used a group of stabilizing...

  7. Plant Phosphoproteomics: Analysis of Plasma Membrane Transporters by Mass Spectrometry

    DEFF Research Database (Denmark)

    Ye, Juanying; Rudashevskaya, Elena; Young, Clifford

      Phosphorylation is a key regulatory factor in all aspects of eukaryotic biology including the regulation of plant membrane-bound transport proteins. To date, mass spectrometry (MS) has been introduced as powerful technology for study of post translational modifications (PTMs), including protein...... phosphorylation. Due to the low abundance of phosphoprotein, the specific enrichment prior to MS analysis is necessary. Plant proton pump (H+-ATPase) is an enzyme controls the major transport processes in the plant, such as root nutrient uptake. Moreover, this pump has been proposed to be involved in other...... important physiological functions, such as stomata aperture, cell elongation, or cellular pH regulation. It is known that the activity of plant plasma membrane H+-ATPase is regulated by phosphorylation. Therefore, we first investigated the phosphorylation profile of plant H+-ATPase by enriching...

  8. Calcium Modulation of Plant Plasma Membrane-Bound Atpase Activities

    Science.gov (United States)

    Caldwell, C.

    1983-01-01

    The kinetic properties of barley enzyme are discussed and compared with those of other plants. Possibilities for calcium transport in the plasma membrane by proton pump and ATPase-dependent calcium pumps are explored. Topics covered include the ph phase of the enzyme; high affinity of barley for calcium; temperature dependence, activation enthalpy, and the types of ATPase catalytic sites. Attention is given to lipids which are both screened and bound by calcium. Studies show that barley has a calmodulin activated ATPase that is found in the presence of magnesium and calcium.

  9. The Zinc-Finger Thylakoid-Membrane Protein FIP Is Involved With Abiotic Stress Response in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Karina L. Lopes

    2018-04-01

    Full Text Available Many plant genes have their expression modulated by stress conditions. Here, we used Arabidopsis FtsH5 protease, which expression is regulated by light stress, as bait in a yeast two-hybrid screen to search for new proteins involved in the stress response. As a result, we found FIP (FtsH5 Interacting Protein, which possesses an amino proximal cleavable transit peptide, a hydrophobic membrane-anchoring region, and a carboxyl proximal C4-type zinc-finger domain. In vivo experiments using FIP fused to green fluorescent protein (GFP showed a plastid localization. This finding was corroborated by chloroplast import assays that showed FIP inserted in the thylakoid membrane. FIP expression was down-regulated in plants exposed to high light intensity, oxidative, salt, and osmotic stresses, whereas mutant plants expressing low levels of FIP were more tolerant to these abiotic stresses. Our data shows a new thylakoid-membrane protein involved with abiotic stress response in Arabidopsis thaliana.

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

    Directory of Open Access Journals (Sweden)

    ARIADNA eGONZÁLEZ-SOLÍS

    2014-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

  12. 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.

  13. 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.

  14. 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

  15. Bicarbonate sulfate exchange in canalicular rat liver plasma membrane vesicles

    Energy Technology Data Exchange (ETDEWEB)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I. (University Hospital, Zurich (Switzerland))

    1987-10-01

    The mechanism(s) and driving forces for biliary excretion of sulfate were investigated in canalicular rat liver plasma membrane vesicles (cLPM). Incubation of cLPM vesicles in the presence of an inside-to-outside (in, out) bicarbonate gradient but not pH or out-to-in sodium gradients, stimulated sulfate uptake 10-fold compared with the absence of bicarbonate and approximately 2-fold above sulfate equilibrium (overshoot). Initial rates of this bicarbonate gradient-driven ({sup 35}S)-sulfate uptake were saturable with increasing concentrations of sulfate and could be inhibited by probenecid, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate, acetazolamide, furosemide, 4-acetamideo-4{prime}-isothiocyanostilbene-2,2{prime}-disulfonic acid, and 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (IC{sub 50}, {approximately}40 {mu}M). Cisinhibition of initial bicarbonate gradient-stimulated sulfate uptake and transstimulation of sulfate uptake in the absence of bicarbonate were observed with sulfate, thiosulfate, and oxalate but not with chloride, nitrate, phosphate, acetate, lactate, glutamate, aspartate, cholate, taurocholate, dehydrocholate, taurodehydrocholate, and reduced or oxidized glutathione. These findings indicate the presence of a sulfate (oxalate)-bicarbonate anion exchange system in canalicular rat liver plasma membranes. These findings support the concept that bicarbonate-sensitive transport system might play an important role in bile acid-independent canalicular bile formation.

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

    Science.gov (United States)

    Bartholomäus, Ingo; Milan-Lobo, Laura; Nicke, Annette; Dutertre, Sébastien; Hastrup, Hanne; Jha, Alok; Gether, Ulrik; Sitte, Harald H; Betz, Heinrich; Eulenburg, Volker

    2008-04-18

    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 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 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.

  17. Bicarbonate sulfate exchange in canalicular rat liver plasma membrane vesicles

    International Nuclear Information System (INIS)

    Meier, P.J.; Valantinas, J.; Hugentobler, G.; Rahm, I.

    1987-01-01

    The mechanism(s) and driving forces for biliary excretion of sulfate were investigated in canalicular rat liver plasma membrane vesicles (cLPM). Incubation of cLPM vesicles in the presence of an inside-to-outside (in, out) bicarbonate gradient but not pH or out-to-in sodium gradients, stimulated sulfate uptake 10-fold compared with the absence of bicarbonate and approximately 2-fold above sulfate equilibrium (overshoot). Initial rates of this bicarbonate gradient-driven [ 35 S]-sulfate uptake were saturable with increasing concentrations of sulfate and could be inhibited by probenecid, N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate, acetazolamide, furosemide, 4-acetamideo-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (IC 50 , ∼40 μM). Cisinhibition of initial bicarbonate gradient-stimulated sulfate uptake and transstimulation of sulfate uptake in the absence of bicarbonate were observed with sulfate, thiosulfate, and oxalate but not with chloride, nitrate, phosphate, acetate, lactate, glutamate, aspartate, cholate, taurocholate, dehydrocholate, taurodehydrocholate, and reduced or oxidized glutathione. These findings indicate the presence of a sulfate (oxalate)-bicarbonate anion exchange system in canalicular rat liver plasma membranes. These findings support the concept that bicarbonate-sensitive transport system might play an important role in bile acid-independent canalicular bile formation

  18. Dynamics of glycine receptor insertion in the neuronal plasma membrane.

    Science.gov (United States)

    Rosenberg, M; Meier, J; Triller, A; Vannier, C

    2001-07-15

    The exocytosis site of newly synthesized glycine receptor was defined by means of a morphological assay to characterize its export from the trans-Golgi Network to the plasma membrane. This was achieved by expressing in transfected neurons an alpha1 subunit bearing an N-terminal tag selectively cleavable from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly occurred at nonsynaptic sites in the cell body and the initial portion of dendrites. At the time of cell surface insertion, the receptors existed as discrete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regions. This localization resulted from diffusion in the plasma membrane and not from exocytosis of transport vesicles directed to dendrites. Kinetic analysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent intermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.

  19. 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.

  20. Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

    Science.gov (United States)

    Fox, Philip D; Haberkorn, Christopher J; Akin, Elizabeth J; Seel, Peter J; Krapf, Diego; Tamkun, Michael M

    2015-06-01

    Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER-plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER-plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER-plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER-plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca(2+) signaling. © 2015. Published by The Company of Biologists Ltd.

  1. 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.

  2. 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.

  3. Closure of Oroantral Communication With Plasma-Rich Fibrin Membrane.

    Science.gov (United States)

    Demetoglu, Umut; Ocak, Hakan; Bilge, Suheyb

    2018-02-26

    Oroantral communication (OAC) is the opening between the maxillary sinus and oral cavity. It may cause oroantral fistula or maxillary sinusitis if left untreated. The surgical closure of the OAC within 48 hours was recommended to avoid the complications like sinus infections. The aim of this study is to evaluate the treatment of OACs with plasma-rich fibrin (PRF) which is safe and easy to implement in the OACs.This study was conducted with the patients, who required the treatment of the OAC, which was developed after the posterior maxillary tooth extraction in the Dental and Maxillofacial Department of the Faculty of Dentistry in Adnan Menderes University.Plasma-rich fibrin membranes were inserted in layers into the tooth socket so that they covered the OAC. Then these membranes were fixated with the sutures to the surrounding gingiva. Antibiotic (amoxicillin/clavulanic acid 1000 mg), analgesic (dexketoprofen trometamol and/or paracetamol), and oral rinse (0.2% chlorhexidine digluconate) agents were prescribed to all patients. The patients were examined in the 3rd and 7th days and 2 months after the operation.All patients tolerated PRF perfectly, and the soft tissue recovery was completed without any problem. Full epithelization was observed in the defect area in all patients. The OAC did not relapse in any patient.Plasma-rich fibrin technique is a simple and effective method, which can be used in the treatment of OACs with a diameter of 5 mm or less with a low risk of complications.

  4. Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

    Science.gov (United States)

    Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

    2017-04-01

    The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

  5. Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H+ -ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum.

    Science.gov (United States)

    Fotopoulos, Vasileios; Holmes, Robert; Hall, J L; Williams, Lorraine E

    2006-01-01

    Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H+ -ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H+ -ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.

  6. Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

    Directory of Open Access Journals (Sweden)

    Leśniewicz Krzysztof

    2012-10-01

    Full Text Available Abstract Background The activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open. Results Bioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed. Conclusions In this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location 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. 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

  9. Towards a membrane proteome in Drosophila: a method for the isolation of plasma membrane

    Directory of Open Access Journals (Sweden)

    Thomas Graham H

    2010-05-01

    Full Text Available Abstract Background The plasma membrane (PM is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP, based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila. Results We show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments. Conclusion A combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this

  10. 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.

  11. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    Science.gov (United States)

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously.

  12. Calcium pumps of plasma membrane and cell interior

    DEFF Research Database (Denmark)

    Strehler, Emanuel E; Treiman, Marek

    2004-01-01

    Calcium entering the cell from the outside or from intracellular organelles eventually must be returned to the extracellular milieu or to intracellular storage organelles. The two major systems capable of pumping Ca2+ against its large concentration gradient out of the cell or into the sarco....../endoplasmatic reticulum are the plasma membrane Ca2+ ATPases (PMCAs) and the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), respectively. In mammals, multigene families code for these Ca2+ pumps and additional isoform subtypes are generated via alternative splicing. PMCA and SERCA isoforms show developmental-, tissue......- and cell type-specific patterns of expression. Different PMCA and SERCA isoforms are characterized by different regulatory and kinetic properties that likely are optimized for the distinct functional tasks fulfilled by each pump in setting resting cytosolic or intra-organellar Ca2+ levels, and in shaping...

  13. Tonoplast- and plasma membrane-localized aquaporin-family transporters in blue hydrangea sepals of aluminum hyperaccumulating plant.

    Directory of Open Access Journals (Sweden)

    Takashi Negishi

    Full Text Available Hydrangea (Hydrangea macrophylla is tolerant of acidic soils in which toxicity generally arises from the presence of the soluble aluminum (Al ion. When hydrangea is cultivated in acidic soil, its resulting blue sepal color is caused by the Al complex formation of anthocyanin. The concentration of vacuolar Al in blue sepal cells can reach levels in excess of approximately 15 mM, suggesting the existence of an Al-transport and/or storage system. However, until now, no Al transporter has been identified in Al hyperaccumulating plants, animals or microorganisms. To identify the transporter being responsible for Al hyperaccumulation, we prepared a cDNA library from blue sepals according to the sepal maturation stage, and then selected candidate genes using a microarray analysis and an in silico study. Here, we identified the vacuolar and plasma membrane-localized Al transporters genes vacuolar Al transporter (VALT and plasma membrane Al transporter 1 (PALT1, respectively, which are both members of the aquaporin family. The localization of each protein was confirmed by the transient co-expression of the genes. Reverse transcription-PCR and immunoblotting results indicated that VALT and PALT1 are highly expressed in sepal tissue. The overexpression of VALT and PALT1 in Arabidopsis thaliana conferred Al-tolerance and Al-sensitivity, respectively.

  14. 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.

  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. pH-induced proton permeability changes of plasma membrane vesicles

    NARCIS (Netherlands)

    Miedema, H; Prins, HBA; Staal, H.

    In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles.

  17. 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 membran...

  18. 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.

  19. 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.

  20. 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.

  1. Remodelling of primary human CD4+ T cell plasma membrane order by n-3 PUFA.

    Science.gov (United States)

    Fan, Yang-Yi; Fuentes, Natividad R; Hou, Tim Y; Barhoumi, Rola; Li, Xian C; Deutz, Nicolaas E P; Engelen, Marielle P K J; McMurray, David N; Chapkin, Robert S

    2018-01-01

    Cell membrane fatty acids influence fundamental properties of the plasma membrane, including membrane fluidity, protein functionality, and lipid raft signalling. Evidence suggests that dietary n-3 PUFA may target the plasma membrane of immune cells by altering plasma membrane lipid dynamics, thereby regulating the attenuation of immune cell activation and suppression of inflammation. As lipid-based immunotherapy might be a promising new clinical strategy for the treatment of inflammatory disorders, we conducted in vitro and in vivo experiments to examine the effects of n-3 PUFA on CD4+ T cell membrane order, mitochondrial bioenergetics and lymphoproliferation. n-3 PUFA were incorporated into human primary CD4+ T cells phospholipids in vitro in a dose-dependent manner, resulting in a reduction in whole cell membrane order, oxidative phosphorylation and proliferation. At higher doses, n-3 PUFA induced unique phase separation in T cell-derived giant plasma membrane vesicles. Similarly, in a short-term human pilot study, supplementation of fish oil (4 g n-3 PUFA/d) for 6 weeks in healthy subjects significantly elevated EPA (20 : 5n-3) levels in CD4+ T cell membrane phospholipids, and reduced membrane lipid order. These results demonstrate that the dynamic reshaping of human CD4+ T cell plasma membrane organisation by n-3 PUFA may modulate down-stream clonal expansion.

  2. 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.

  3. Serial plasma concentrations of atrial natriuretic peptide, plasma renin activity, aldosterone, and antidiuretic hormone in neonates on extracorporeal membrane oxygenation.

    NARCIS (Netherlands)

    Semmekrot, B.A.; Pesman, G.J.; Span, P.N.; Sweep, C.G.J.; Heyst, A.F.J. van; Monnens, L.A.H.; Bor, M. van de; Tanke, R.B.; Staak, F.H.J.M. van der

    2002-01-01

    To obtain information on water and salt regulating hormones and volume homeostasis during neonatal extracorporeal membrane oxygenation (ECMO), serial determinations of atrial natriuretic peptide (ANP), plasma renin activity (PRA), aldosterone (Aldo), antidiuretic hormone (ADH), colloid-osmotic

  4. Red wine activates plasma membrane redox system in human erythrocytes.

    Science.gov (United States)

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-01-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.

  5. 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.

  6. Preparation and characteristics of acrylic acid/styrene composite plasma polymerized membranes

    Science.gov (United States)

    Jiang, Zhongqing; Jiang, Zhong-jie; Shi, Yicai; Meng, Yuedong

    2010-08-01

    Plasma polymerization has gained increasing interest for the deposition of functional plasma-polymerized membranes suitable for a wide range of applications on account of its advantageous features. In this work, acrylic acid/styrene composite plasma polymerized membranes were synthesized by plasma polymerization of a mixture of acrylic acid and styrene monomers in a low-frequency after-glow capacitively coupled plasma (CCP) discharge process. The structure and composition of the plasma polymerized membranes were characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The results showed that the partial pressure ratio between acrylic acid (AA) and styrene (St), applied discharge power and the energy of the extracted particles have considerable effects on the structure and the content of functional groups of the deposited membranes.

  7. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

    DEFF Research Database (Denmark)

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael

    2010-01-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood...... for Rho1 and Rho2, in the regulation of sphingolipid metabolism....

  16. 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

  17. Developmental distribution of the plasma membrane-enriched proteome in the maize primary root growth zone

    Directory of Open Access Journals (Sweden)

    Zhe eZhang

    2013-03-01

    Full Text Available Within the growth zone of the maize primary root, there are well-defined patterns of spatial and temporal organization of cell division and elongation. However, the processes underlying this organization remain poorly understood. To gain additional insights into the differences amongst the defined regions, we performed a proteomic analysis focusing on fractions enriched for plasma membrane (PM proteins. The PM is the interface between the plant cell and the apoplast and/or extracellular space. As such, it is a key structure involved in the exchange of nutrients and other molecules as well as in the integration of signals that regulate growth and development. Despite the important functions of PM-localized proteins in mediating these processes, a full understanding of dynamic changes in PM proteomes is often impeded by low relative concentrations relative to total proteins. Using a relatively simple strategy of treating microsomal fractions with Brij-58 detergent to enrich for PM proteins, we compared the developmental distribution of proteins within the root growth zone which revealed a number of previously known as well as novel proteins with interesting patterns of abundance. For instance, the quantitative proteomic analysis detected a gradient of PM aquaporin proteins similar to that previously reported using immunoblot analyses, confirming the veracity of this strategy. Cellulose synthases increased in abundance with increasing distance from the root apex, consistent with expected locations of cell wall deposition. The similar distribution pattern for Brittle-stalk-2-like protein 3 implicate that this protein may also have cell wall related functions. These results show that the simplified PM enrichment method previously demonstrated in Arabidopsis can be successfully applied to completely unrelated plant tissues and provide insights into differences in the PM proteome throughout growth and development zones of the maize primary root.

  18. Tailoring the properties of asymmetric cellulose acetate membranes by gas plasma etching.

    Science.gov (United States)

    Olde Riekerink, M B; Engbers, G H M; Wessling, M; Feijen, J

    2002-01-15

    Cellulose triacetate (CTA) ultrafilters and cellulose acetate blend (CAB) desalination membranes were treated with a radiofrequency gas plasma (tetrafluoromethane (CF(4)) or carbon dioxide (CO(2)), 47-49 W, 0.04-0.08 mbar). Treatment times were varied between 15 s and 120 min. The plasma-treated top layer of the membranes was characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and contact angle measurements to obtain information about surface structure, chemistry, and wettability, respectively. The membrane properties (e.g., permeability, selectivity, fouling) were studied by waterflux measurements, molecular weight cutoff measurements, and fouling experiments with bovine serum albumin. CO(2) plasma treatment resulted in gradual etching of the membrane's dense top layer. Permeation and selectivity changed significantly for treatment times of 0-15 min for CTA and 5-60 min for CAB membranes. Moreover, CTA membranes were hydrophilized during CO(2) plasma treatment whereas CF(4) plasma treatment led to hydrophobic surfaces due to strong fluorination of the top layer. This study shows that gas plasma etching can tailor the properties of asymmetric cellulose acetate membranes by simultaneously modifying the chemistry and structure of the top layer. The low fouling properties of CTA membranes were thereby largely maintained.

  19. Alterations in lipid composition and fluidity of liver plasma membranes in copper-deficient rats

    Energy Technology Data Exchange (ETDEWEB)

    Lei, K.Y.; Rosenstein, F.; Shi, F.; Hassel, C.A.; Carr, T.P.; Zhang, J. (Univ. of Arizona, Tucson (USA))

    1988-07-01

    In view of the importance of membrane fluidity on cell functions, the influence of phospholipid acyl groups on membrane fluidity, and the changes in lipid metabolism induced by copper (Cu) deficiency, this study was designed to examine the influence of dietary Cu on the lipid composition and fluidity of liver plasma membranes. Male Sprague-Dawley rats were divided into two dietary treatments, namely Cu deficient and Cu adequate. After 8 weeks of treatment, liver plasma membranes were isolated by sucrose density gradient centrifugation. The lipid fluidity of plasma membranes, as assessed by the intramolecular eximer fluorescence of 1,3-di(1-pyrenyl) propane, was significantly depressed by Cu deficiency. In addition, Cu deficiency significantly reduced the content of arachidonic and palmitoleic acids but increased the docosatetraenoic acids of membrane phospholipids. This alteration in unsaturated phospholipid fatty acid composition, especially the large reduction in arachidonic acid, may have contributed to the depressed membrane fluidity. Furthermore, Cu deficiency also markedly altered the fatty acid composition of the triacylglycerols associated with the plasma membranes. Thus, the lipid composition and fluidity of liver plasma membranes are responsive to the animal's Cu status.

  20. A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs).

    Science.gov (United States)

    Lanigan, Peter M P; Ninkovic, Tanja; Chan, Karen; de Mello, Andrew J; Willison, Keith R; Klug, David R; Templer, Richard H; Neil, Mark A A; Ces, Oscar

    2009-04-21

    We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

  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. 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

  3. 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.

  4. Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane.

    Directory of Open Access Journals (Sweden)

    Laura Paparelli

    2016-09-01

    Full Text Available Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH. We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided.

  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. 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.

  7. Vesicle-associated membrane protein 2 mediates trafficking of {alpha}5{beta}1 integrin to the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Hasan, Nazarul [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States); Hu, Chuan, E-mail: chuan.hu@louisville.edu [Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 319 Abraham Flexner Way, Room 515, Louisville, KY 40202 (United States)

    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 {alpha}5{beta}1 integrin. VAMP2 was present on vesicles containing endocytosed {beta}1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface {alpha}5{beta}1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of {alpha}5{beta}1, without altering cell surface expression of {alpha}2{beta}1 integrin or {alpha}3{beta}1 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 {alpha}5{beta}1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.

  8. Deletion analysis of AGD1 reveals domains crucial for plasma membrane recruitment and function in root hair polarity.

    Science.gov (United States)

    Yoo, Cheol-Min; Naramoto, Satoshi; Sparks, J Alan; Khan, Bibi Rafeiza; Nakashima, Jin; Fukuda, Hiroo; Blancaflor, Elison B

    2018-01-29

    AGD1, a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP), functions in specifying root hair polarity in Arabidopsis thaliana To better understand how AGD1 modulates root hair growth, we generated full-length and domain-deleted AGD1-green fluorescent protein (GFP) constructs, and followed their localization during root hair development. AGD1-GFP localized to the cytoplasm and was recruited to specific regions of the root hair plasma membrane (PM). Distinct PM AGD1-GFP signal was first detected along the site of root hair bulge formation. The construct continued to mark the PM at the root hair apical dome, but only during periods of reduced growth. During rapid tip growth, AGD1-GFP labeled the PM of the lateral flanks and dissipated from the apical-most PM. Deletion analysis and a single domain GFP fusion revealed that the pleckstrin homology (PH) domain is the minimal unit required for recruitment of AGD1 to the PM. Our results indicate that differential recruitment of AGD1 to specific PM domains is an essential component of the membrane trafficking machinery that facilitates root hair developmental phase transitions and responses to changes in the root microenvironment. © 2018. Published by The Company of Biologists Ltd.

  9. Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.

    Science.gov (United States)

    Takahashi, Daisuke; Kawamura, Yukio; Yamashita, Tetsuro; Uemura, Matsuo

    2012-03-02

    The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC-MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (Arabidopsis and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells.

  10. 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.

  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. Application of Higher Density Iron Oxide Nanoparticle Pellicles to Enrich the Plasma Membrane and Its Proteome from Cells in Suspension.

    Science.gov (United States)

    Rose, Rebecca L; Choksawangkarn, Waeowalee; Fenselau, Catherine

    2018-01-01

    Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.

  13. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

    Science.gov (United States)

    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © 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.

  14. 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.

  15. The influence of hemodialysis membranes on the plasma insulin level of diabetic patients on maintenance hemodialysis.

    Science.gov (United States)

    Abe, M; Kikuchi, F; Kaizu, K; Matsumoto, K

    2008-05-01

    The aim of the present study was to evaluate the alteration in plasma immunoreactive insulin (IRI) concentrations due to hemodialysis (HD) treatment by using three types of membranes in diabetic HD patients. We recruited 20 outpatients on maintenance HD with diabetes for this crossover study. HD was performed using membranes made of cellulose triacetate (CTA), polyester-polymer alloy (PEPA), and polysulphone (PS). These membranes were used for 2 weeks (6 HD sessions) in each patient in a randomized order decided by drawing lots. Blood samples were obtained at the beginning and end of the HD session from the blood tubing at the arterial (A) site. At 60 min after the initiation of dialysis, blood samples were obtained from the blood tubing at both the A and venous (V) sites of the dialyzer. The plasma IRI levels decreased significantly at the sites an hour after initiating HD in all membranes. The clearance of IRI was significantly higher in the case of the PS membrane when compared with the CTA and PEPA membranes. It was concluded that plasma insulin is cleared by HD, and the rate differs for each membrane. Plasma insulin clearance with the PS membrane is higher than that with the PEPA and CTA membranes.

  16. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    International Nuclear Information System (INIS)

    Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

    2010-01-01

    Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

  17. 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.

  18. Uncovering Arabidopsis membrane protein interactome enriched in transporters using mating-based split ubiquitin assays and classification models

    Directory of Open Access Journals (Sweden)

    Jin eChen

    2012-06-01

    Full Text Available High-throughput data are a double-edged sword; for the benefit of large amount of data, there is an associated cost of noise. To increase reliability and scalability of high-throughput protein interaction data generation, we tested the efficacy of classification to enrich potential protein-protein interactions (pPPIs. We applied this method to identify interactions among Arabidopsis membrane proteins enriched in transporters. We validated our method with multiple retests. Classification improved the quality of the ensuing interaction network and was effective in reducing the search space and increasing true positive rate. The final network of 541 interactions among 239 proteins (of which 179 are transporters is the first protein interaction network enriched in membrane transporters reported for any organism. This network has similar topological attributes to other published protein interaction networks. It also extends and fills gaps in currently available biological networks in plants and allows building a number of hypotheses about processes and mechanisms involving signal-transduction and transport systems.

  19. Specific and efficient targeting of cyanobacterial bicarbonate transporters to the inner envelope membrane of chloroplasts in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Susumu eUehara

    2016-02-01

    Full Text Available Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

  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. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

    Czech Academy of Sciences Publication Activity Database

    Irani, N. G.; Di Rubbo, S.; Mylle, E.; Van den Begin, J.; Schneider-Pizon, J.; Hniličková, Jaroslava; Šíša, Miroslav; Buyst, D.; Vilarrasa-Blasi, J.; Szatmári, A. M.; Van Damme, D.; Mishev, K.; Codreanu, M. C.; Kohout, Ladislav; Strnad, Miroslav; Cano-Delgado, A. I.; Friml, J.; Madder, A.; Russinova, E.

    2012-01-01

    Roč. 8, č. 6 (2012), s. 583-589 ISSN 1552-4450 R&D Projects: GA MŠk 1M06030 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50380511 Keywords : receptor kinase BRI1 * steroid-binding protein-1 * plant-cells * brassinosteroid biosynthesis * dependent endocytosis * auxin transport * Arabidopsis Subject RIV: CC - Organic Chemistry Impact factor: 12.948, year: 2012

  2. 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.

  3. Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

    Directory of Open Access Journals (Sweden)

    L.S.L.S. Reis

    2016-06-01

    Full Text Available ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion, scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342. The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

  4. 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.

  5. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

    Science.gov (United States)

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua

    2015-12-04

    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. LPS-Induced Macrophage Activation and Plasma Membrane Fluidity Changes are Inhibited Under Oxidative Stress.

    Science.gov (United States)

    de la Haba, Carlos; Morros, Antoni; Martínez, Paz; Palacio, José R

    2016-12-01

    Macrophage activation is essential for a correct and efficient response of innate immunity. During oxidative stress membrane receptors and/or membrane lipid dynamics can be altered, leading to dysfunctional cell responses. Our aim is to analyze membrane fluidity modifications and cell function under oxidative stress in LPS-activated macrophages. Membrane fluidity of individual living THP-1 macrophages was evaluated by the technique two-photon microscopy. LPS-activated macrophage function was determined by TNFα secretion. It was shown that LPS activation causes fluidification of macrophage plasma membrane and production of TNFα. However, oxidative stress induces rigidification of macrophage plasma membrane and inhibition of cell activation, which is evidenced by a decrease of TNFα secretion. Thus, under oxidative conditions macrophage proinflammatory response might develop in an inefficient manner.

  7. 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.

  8. Imaging of blood plasma coagulation at supported lipid membranes.

    Science.gov (United States)

    Faxälv, Lars; Hume, Jasmin; Kasemo, Bengt; Svedhem, Sofia

    2011-12-15

    The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments

    Directory of Open Access Journals (Sweden)

    Kuismanen Esa

    2005-05-01

    Full Text Available Abstract Background Membrane fusion requires the formation of a complex between a vesicle protein (v-SNARE and the target membrane proteins (t-SNAREs. Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3. Results Endogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM, which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures. Conclusion Our data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function.

  10. EFFECT OF HIGH PH ON THE PLASMA-MEMBRANE POTENTIAL AND CONDUCTANCE IN ELODEA-DENSA

    NARCIS (Netherlands)

    MIEDEMA, H; FELLE, H; PRINS, HBA

    In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the

  11. 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

    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...

  12. 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

  13. 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

  14. Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes.

    Science.gov (United States)

    Eubel, Holger; Meyer, Etienne H; Taylor, Nicolas L; Bussell, John D; O'Toole, Nicholas; Heazlewood, Joshua L; Castleden, Ian; Small, Ian D; Smith, Steven M; Millar, A Harvey

    2008-12-01

    Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, beta-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a

  15. The A/ENTH Domain-Containing Protein AtECA4 Is an Adaptor Protein Involved in Cargo Recycling from the trans-Golgi Network/Early Endosome to the Plasma Membrane.

    Science.gov (United States)

    Nguyen, Hong Hanh; Lee, Myoung Hui; Song, Kyungyoung; Ahn, Gyeongik; Lee, Jihyeong; Hwang, Inhwan

    2018-01-06

    Endocytosis and subsequent trafficking pathways are crucial for regulating the activity of plasma membrane-localized proteins. Depending on cellular and physiological conditions, the internalized cargoes are sorted at (and transported from) the trans-Golgi network/early endosome (TGN/EE) to the vacuole for degradation or recycled back to the plasma membrane. How this occurs at the molecular level remains largely elusive. Here, we provide evidence that the ENTH domain-containing protein AtECA4 plays a crucial role in recycling cargoes from the TGN/EE to the plasma membrane in Arabidopsis thaliana. AtECA4:sGFP primarily localized to the TGN/EE and plasma membrane (at low levels). Upon NaCl or mannitol treatment, AtECA4:sGFP accumulated at the TGN/EE at an early time point but was released from the TGN/EE to the cytosol at later time points. The ateca4 mutant showed higher resistance to osmotic stress and more sensitive to exogenous abscisic acid (ABA) than the wild type, as well as increased expression of ABA-inducible genes RD29A and RD29B. Consistently, ABCG25, a plasma membrane-localized ABA exporter, accumulated at the prevacuolar compartment in ateca4, indicating a defect in recycling to the plasma membrane. However, the role of AtECA4 in cargo recycling is not specific to ABCG25, as it also functions in the recycling of BRI1. These results suggest that AtECA4 plays a crucial role in the recycling of endocytosed cargoes from the TGN/EE to the plasma membrane. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

  16. Members of rice plasma membrane intrinsic proteins subfamily are involved in arsenite permeability and tolerance in plants.

    Science.gov (United States)

    Mosa, Kareem A; Kumar, Kundan; Chhikara, Sudesh; Mcdermott, Joseph; Liu, Zijuan; Musante, Craig; White, Jason C; Dhankher, Om Parkash

    2012-12-01

    Rice accumulates high level of arsenic (As) in its edible parts and thus plays an important role in the transfer of As into the food chain. However, the mechanisms of As uptake and its detoxification in rice are not well understood. Recently, members of the Nodulin 26-like intrinsic protein (NIP) subfamily of plant aquaporins were shown to transport arsenite in rice and Arabidopsis. Here we report that members of the rice plasma membrane intrinsic protein (PIP) subfamily are also involved in As tolerance and transport. Based on the homology search with the mammalian AQP9 and yeast Fps1 arsenite transporters, we identified and cloned five rice PIP gene subfamily members. qRT-PCR analysis of PIPs in rice root and shoot tissues revealed a significant down regulation of transcripts encoding OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;6, and OsPIP2;7 in response to arsenite treatment. Heterologous expression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Xenopus laevis oocytes significantly increased the uptake of arsenite. Overexpression of OsPIP2;4, OsPIP2;6, and OsPIP2;7 in Arabidopsis yielded enhanced arsenite tolerance and higher biomass accumulation. Further, these transgenic plants showed no significant accumulation of As in shoot and root tissues in long term uptake assays. Whereas, short duration exposure to arsenite caused both active influx and efflux of As in the roots. The data suggests a bidirectional arsenite permeability of rice PIPs in plants. These rice PIPs genes will be highly useful for engineering important food and biofuel crops for enhanced crop productivity on contaminated soils without increasing the accumulation of toxic As in the biomass or edible tissues.

  17. Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth.

    Science.gov (United States)

    Wang, Yin; Noguchi, Ko; Ono, Natsuko; Inoue, Shin-ichiro; Terashima, Ichiro; Kinoshita, Toshinori

    2014-01-07

    Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO2, and the plant hormone abscisic acid. Light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H(+)-ATPase, and plasma membrane inward-rectifying K(+) channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO2 uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H(+)-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with ∼42-63% greater fresh and dry weights than the wild type in the first 25 d of growth. The dry weights of total flowering stems of 45-d-old transgenic plants, including seeds, siliques, and flowers, were ∼36-41% greater than those of the wild type. In addition, stomata in the transgenic plants closed normally in response to darkness and abscisic acid. In contrast, the overexpression of phototropin or inward-rectifying K(+) channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H(+)-ATPase in guard cells is useful for the promotion of plant growth.

  18. 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...

  19. Tailoring the properties of asymmetric cellulose acetate membranes by gas plasma etching

    NARCIS (Netherlands)

    Olde riekerink, M.B.; Engbers, G.H.M.; Wessling, Matthias; Feijen, Jan

    2002-01-01

    Cellulose triacetate (CTA) ultrafilters and cellulose acetate blend (CAB) desalination membranes were treated with a radiofrequency gas plasma (tetrafluoromethane (CF4) or carbon dioxide (CO2), 47¿49 W, 0.04¿0.08 mbar). Treatment times were varied between 15 s and 120 min. The plasma-treated top

  20. Characterization of the intracellular and the plasma membrane Ca2+-ATPases in fractionated pig brain membranes using calcium pump inhibitors.

    Science.gov (United States)

    Salvador, J M; Mata, A M

    1998-03-15

    The Ca2+-ATPase activity of isolated membranes and purified plasma membrane ATPase from pig brain was measured in the presence of specific inhibitors. The inhibition of the enzymatic activity by vanadate presents a lower affinity in microsomes than in the synaptic plasma membrane vesicles, showing K0.5 of 0.4 and 0.2 microM, respectively. The purified enzyme showed a higher sensitivity to vanadate with a K0.5 of 0.10 microM. Thapsigargin (Tg) and 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) were stronger inhibitors of the Ca2+-ATPase activity in microsomes than in the synaptic membrane vesicles. The activity of the purified enzyme was not affected by Tg and only partially by BHQ. Cyclopiazonic acid inhibited the enzymatic activity in all fractions, being more sensitive in microsomes. The microsome preparation incorporated 32P from [gamma-32P]ATP into two main proteins that appear at approx 110,000 and 140,000. According to the inhibition pattern, the lower phosphorylated band was identified as the sarco(endo)plasmic reticulum Ca2+-ATPase, being in a higher percentage than the upper band. Synaptic membrane vesicles also incorporated radioactive 32P into two protein bands. The 140,000 protein (upper band) shows the typical behavior of the purified plasma membrane Ca2+-ATPase, being more abundant in this preparation than the organellar Ca2+-pump (lower band). This study highlights the heterogeneous nature of the Ca2+-ATPase activity measured in brain membrane fractions. Copyright 1998 Academic Press.

  1. Lactoperoxidase-Catalyzed Iodination of Plasmamembrane Lipids and Proteins in Arabidopsis Protoplasts

    OpenAIRE

    Merle M., Millard; Plant Stress Laboratory, Agricultural Research Service, U. S. Department of Agriculture

    1988-01-01

    Lactoperoxidase-catalyzed iodination was evaluated as a technique to identify plasma membrane components in protoplasts isolated from leaf tissue of Arabidopsis thaliana (L.) Heynh. Restriction of the probe to the plasma membrane was assessed by determining radioactivity in lipids characteristic of intracellular organelles after labeling intact versus broken protoplasts. In-tact protoplasts were purified on a sucrose-sorbitol gradient and iodinated using Na[^I] with lactoperoxidase and H_2O_2...

  2. INTERACTION OF HYDROGEN VHF – PLASMA WITH PD – MEMBRANE

    OpenAIRE

    Savitsky, A. A.; Pankov, V. V.

    2013-01-01

    Atomic hydrogen interaction with metals processes introduces major interest concern for atomic and hydrogen energetic, and also space materials technology. In this paper the data on permeability of hydrogen through palladium membranes in a wide temperature range are cited, and also is informed about the effect of atomic hydrogen superpermeability through palladium membranes

  3. Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

    Science.gov (United States)

    Koike, Michiya; Sutoh, Keita; Kawakami, Akira; Torada, Atsushi; Oono, Kiyoharu; Imai, Ryozo

    2005-12-01

    As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

  4. 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....

  5. 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...

  6. Emerging role of chemoprotective agents in the dynamic shaping of plasma membrane organization.

    Science.gov (United States)

    Fuentes, Natividad R; Salinas, Michael L; Kim, Eunjoo; Chapkin, Robert S

    2017-09-01

    In the context of an organism, epithelial cells by nature are designed to be the defining barrier between self and the outside world. This is especially true for the epithelial cells that form the lining of the digestive tract, which absorb nutrients and serve as a barrier against harmful substances. These cells are constantly bathed by a complex mixture of endogenous (bile acids, mucus, microbial metabolites) and exogenous (food, nutrients, drugs) bioactive compounds. From a cell biology perspective, this type of exposure would directly impact the plasma membrane, which consists of a myriad of complex lipids and proteins. The plasma membrane not only functions as a barrier but also as the medium in which cellular signaling complexes form and function. This property is mediated by the organization of the plasma membrane, which is exquisitely temporally (nanoseconds to minutes) and spatially (nanometers to micrometers) regulated. Since numerous bioactive compounds found in the intestinal lumen can directly interact with lipid membranes, we hypothesize that the dynamic reshaping of plasma membrane organization underlies the chemoprotective effect of select membrane targeted dietary bioactives (MTDBs). This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. Suicidal behaviour is associated with decreased esterified cholesterol in plasma and membrane fluidity of platelets.

    Science.gov (United States)

    Mathew, Boby; Srinivasan, Krishnamachari; Pradeep, Johnson; Thomas, Tinku; Mandal, Amit Kumar

    2018-02-01

    Altered cholesterol levels in body fluids and brain tissues have been shown to be associated with suicidal behaviour, violence and aggression. But the biological underpinnings of this association in the pathophysiology of suicide are not clear. Cholesterol plays a crucial role in maintaining the cellular membrane fluidity and alterations in cellular membrane fluidity may impair serotonergic neurotransmission in the central nervous system. We measured plasma esterified cholesterol and platelet membrane fluidity using fluorescence anisotropy and estimated flow activation energy which is a measure of order of membrane lipid bilayer in patients with recent suicidal attempt and compared with age and gender matched controls. The plasma esterified cholesterol, platelet membrane fluidity and flow activation energy was found to be significantly lower in patients with recent suicidal attempts compared to controls. Altered levels of plasma esterified cholesterol which is in equilibrium with membrane cholesterol might have resulted in decreased membrane fluidity and an increase in the order of membrane lipid bilayer. This might impair the serotonergic neurotransmission, which has been implicated in the pathophysiology of suicide. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Secretion Trap Tagging of Secreted and Membrane-Spanning Proteins Using Arabidopsis Gene Traps

    Science.gov (United States)

    Andrew T. Groover; Joseph R. Fontana; Juana M. Arroyo; Cristina Yordan; W. Richard McCombie; Robert A. Martienssen

    2003-01-01

    Secreted and membrane-spanning proteins play fundamental roles in plant development but pose challenges for genetic identification and characterization. We describe a "secretion trap" screen for gene trap insertions in genes encoding proteins routed through the secretory pathway. The gene trap transposon encodes a ß-glucuronidase reporter enzyme...

  14. Reduced Biosynthesis of Digalactosyldiacylglycerol, a Major Chloroplast Membrane Lipid, Leads to Oxylipin Overproduction and Phloem Cap Lignification in Arabidopsis.

    Science.gov (United States)

    Lin, Yang-Tsung; Chen, Lih-Jen; Herrfurth, Cornelia; Feussner, Ivo; Li, Hsou-Min

    2016-01-01

    DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA. © 2016 American Society of Plant Biologists. All rights reserved.

  15. The plasma membrane as a capacitor for energy and metabolism

    Science.gov (United States)

    Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini

    2016-01-01

    When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as “capacitors for energy and metabolism.” Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520

  16. 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

  17. Does Increased Expression of the Plasma Membrane Calcium-ATPase Isoform 2 Confer Resistance to Apoptosis on Breast Cancer Cells?

    National Research Council Canada - National Science Library

    VanHouten, Joshua N

    2008-01-01

    The plasma membrane calcium ATPase isoform 2 (PMCA2) is highly expressed on the apical membrane of mammary epithelial cells during lactation, and is the predominant pump responsible for calcium transport into milk...

  18. Free-cholesterol loading does not trigger phase separation of the fluorescent sterol dehydroergosterol in the plasma membrane of macrophages

    DEFF Research Database (Denmark)

    Wüstner, Daniel

    2008-01-01

    membrane distribution of the fluorescent cholesterol-mimicking sterol dehydroergosterol (DHE) was investigated in FC-loaded J774 macrophages. Wide field fluorescence and deconvolution microscopy were combined with quantitative assessment of sterol distribution in straightened plasma membrane image segments...

  19. 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.

  20. 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

    2016-07-01

    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.

  1. Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

    Directory of Open Access Journals (Sweden)

    Sokolowska Izabela

    2012-08-01

    Full Text Available Abstract Liver infection with hepatitis B virus (HBV, a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (− and differentiated (+ cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (− and (+ cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.

  2. 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

  3. Fluorinated carboxylic membranes deposited by plasma enhanced chemical vapour deposition for fuel cell applications

    Energy Technology Data Exchange (ETDEWEB)

    Thery, J.; Faucheux, V.; Truffier-Boutry, D.; Martinent, A.; Laurent, J.-Y. [Laboratory of Printed Component, LITEN, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 09 (France); Martin, S.; Le Van Jodin, L. [Laboratory of Components for the Micro-storage of Energy, LITEN, CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 09 (France)

    2010-09-01

    Among the fuel cell technologies, the polymer electrolyte membrane fuel cells (PEMFCs) are particularly promising because they are energy-efficient, clean, and fuel-flexible (i.e., can use hydrogen or methanol). The great majority of PEM fuel cells rely on a polymer electrolyte from the family of perfluorosulfonic acid membranes, nevertheless alternative materials are currently being developed, mainly to offer the alternative workout techniques which are required for the portable energy sources. Plasma polymerization represents a good solution, as it offers the possibility to deposit thin layer with an accurate and homogeneous thickness, even on 3D surfaces. In this paper, we present the results for the growth of proton conductive fluoro carboxylic membranes elaborated by plasma enhanced chemical vapour deposition. These membranes present conductivity values of the same order than the one of Nafion {sup registered}. The properties of the membrane, such as the chemical composition, the ionic conductivity, the swelling behaviour and the permeability were correlated to the plasma process parameters. The membranes were integrated in fuel cells on porous substrates and we present here the results regarding the barrier effect and the power output. Barrier effect similar to those of 40 {mu}m Nafion {sup registered} layers was reached for 10 {mu}m thick carboxylic membranes. Power outputs around 3 mW cm{sup -2} were measured. We discuss the results regarding the gas barrier effect and the power outputs. (author)

  4. 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.

  5. Fluorinated carboxylic membranes deposited by plasma enhanced chemical vapour deposition for fuel cell applications

    Science.gov (United States)

    Thery, J.; Martin, S.; Faucheux, V.; Le Van Jodin, L.; Truffier-Boutry, D.; Martinent, A.; Laurent, J.-Y.

    Among the fuel cell technologies, the polymer electrolyte membrane fuel cells (PEMFCs) are particularly promising because they are energy-efficient, clean, and fuel-flexible (i.e., can use hydrogen or methanol). The great majority of PEM fuel cells rely on a polymer electrolyte from the family of perfluorosulfonic acid membranes, nevertheless alternative materials are currently being developed, mainly to offer the alternative workout techniques which are required for the portable energy sources. Plasma polymerization represents a good solution, as it offers the possibility to deposit thin layer with an accurate and homogeneous thickness, even on 3D surfaces. In this paper, we present the results for the growth of proton conductive fluoro carboxylic membranes elaborated by plasma enhanced chemical vapour deposition. These membranes present conductivity values of the same order than the one of Nafion ®. The properties of the membrane, such as the chemical composition, the ionic conductivity, the swelling behaviour and the permeability were correlated to the plasma process parameters. The membranes were integrated in fuel cells on porous substrates and we present here the results regarding the barrier effect and the power output. Barrier effect similar to those of 40 μm Nafion ® layers was reached for 10 μm thick carboxylic membranes. Power outputs around 3 mW cm -2 were measured. We discuss the results regarding the gas barrier effect and the power outputs.

  6. Reference: 538 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available d A et al. 2007 Apr. Plant Physiol. 143(4):1576-89. Membrane proteins within the sieve element-companion cel...RS6, selected from hybridomas raised against sieve elements isolated from Califor...psis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve... elements, but not companion cells, and accumulates at the earliest stages of sieve...ch domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve

  7. 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.

  8. 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

    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...... of this research is to improve individualized treatment and increase cancer patient survival, further development is crucial to direct analysis of clinically relevant patient samples. These efforts include optimized specimen handling and preparation as well as improved proteomics platforms. Identification......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...

  9. 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

  10. Surface Modification of Polyvinylidene Fluoride (PVDF) Membranes by Low-Temperature Plasma with Grafting Styrene

    Science.gov (United States)

    Chen, Jian; Li, Jiding; Chen, Cuixian

    2009-02-01

    In order to control the surface pore sizes of polyvinylidene fluoride membranes and their distribution, low temperature plasma-induced grafting modifications of PVDF were studied to prepare hydrophobe membranes. By argon (Ar) treating and subsequent grafting reaction, a hydrophobe monomer, styrene, was introduced into the PVDF membrane. Fourier transform infrared attenuated total reflection (FTIR-ATR) was utilized to characterize the chemical and physical changes in the Ar plasma modified membrane. The surface modifications of PVDF membranes were investigated by using scanning electron microscopy (SEM), atomic force microscopy (AFM) and differential scanning calorimeter (DSC). The water permeability and the solute rejection were measured by PVDF membrane modified in different graft conditions. Results demonstrated that the pores in the modified membranes get smaller and the distribution of pores gets narrowed with the increase in grafting reaction duration. Longer graft time caused the water flux of PVDF membrane to decrease from 578 kg/(m2·h) to 23 kg/(m2·h) and the solute rejection to increase from 73% to 92%.

  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. Effect of gamma-radiation on the plasma and vacuolar membranes of cultured spinach cells

    International Nuclear Information System (INIS)

    Mitsuhashi, N.; Koshiba, T.; Sato, M.

    1998-01-01

    By the use of 31 P NMR spectroscopy, a higher radiosensitivity of the vacuolar membrane (tonoplast) than the plasma membrane was inferred for cultured spinach cells irradiated with 0-7.5 kGy and immediately transferred to fresh medium. In non-irradiated cells, inorganic phosphate (Pi) was rapidly taken up from the medium and transported into the vacuole; the content of cytoplasmic Pi was maintained at a constant level throughout the incubation period (7 days), whilst the Pi in the vacuole was exhausted. In irradiated cells, Pi uptake was not greatly altered up to 5 kGy, and fluorescein diacetate, a substance used to determine the integrity of the plasma membrane, was readily absorbed. However, movement of the Pi taken up into the vacuole across the tonoplast was markedly reduced with increasing dose. This membrane impairment was not amplified during subsequent incubation. A crucial question as to the fate of the Pi is critically discussed. (author)

  13. 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

  14. ABBA regulates plasma-membrane and actin dynamics to promote radial glia extension.

    Science.gov (United States)

    Saarikangas, Juha; Hakanen, Janne; Mattila, Pieta K; Grumet, Martin; Salminen, Marjo; Lappalainen, Pekka

    2008-05-01

    Radial glia play key roles in neuronal migration, axon guidance, and neurogenesis during development of the central nervous system. However, the molecular mechanisms regulating growth and morphology of these extended cells are unknown. We show that ABBA, a novel member of the IRSp53-MIM protein family, is enriched in different types of radial glia. ABBA binds ATP-actin monomers with high affinity and deforms PtdIns(4,5)P(2)-rich membranes in vitro through its WH2 and IM domains, respectively. In radial-glia-like C6-R cells, ABBA localises to the interface between the actin cytoskeleton and plasma membrane, and its depletion by RNAi led to defects in lamellipodial dynamics and process extension. Together, this study identifies ABBA as a novel regulator of actin and plasma membrane dynamics in radial glial cells, and provides evidence that membrane binding and deformation activity is critical for the cellular functions of IRSp53-MIM-ABBA family proteins.

  15. 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.

  16. 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.

  17. Plasma-induced Styrene Grafting onto the Surface of Polytetrafluoroethylene Powder for Proton Exchange Membrane Application

    Science.gov (United States)

    Lan, Yan; Cheng, Cheng; Zhang, Suzhen; Ni, Guohua; Chen, Longwei; Yang, Guangjie; Nagatsu, M.; Meng, Yuedong

    2011-10-01

    Low-temperature plasma treatment was adopted to graft styrene onto polytetrafluoroethylene (PTFE) powder, which is widely used in the fabrication of proton exchange membrane (PEM). The grafted PTFE powder was sulfonated in chlorosulfonic acid and fabricated into a membrane, which was used as inexpensive PEM material for a proton exchange membrane fuel cell (PEMFC). Fourier transform infrared spectroscopy attenuated total reflection spectroscopy (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analysis were used to characterize the structure of the sulfonated PTFE powder. The results showed that all the PTFE powders were successfully grafted by nitrogen plasma and then sulfonated under such experimental conditions. A scanning electron microscopy (SEM) image indicated that the fabricated membrane exhibits flat morphology and homogenous structure. The ion exchange capacity (IEC) of this kind of PEM was also investigated.

  18. Duration of ultrasound-mediated enhanced plasma membrane permeability

    NARCIS (Netherlands)

    Lammertink, Bart; Deckers, Roel; Storm, Gerrit; Moonen, Chrit; Bos, Clemens

    2015-01-01

    Ultrasound (US) induced cavitation can be used to enhance the intracellular delivery of drugs by transiently increasing the cell membrane permeability. The duration of this increased permeability, termed temporal window, has not been fully elucidated. In this study, the temporal window was

  19. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in sig...

  20. Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

    Science.gov (United States)

    Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

    2016-09-01

    Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

  1. Cellulose microfibril deposition: coordinated activity at the plant plasma membrane

    NARCIS (Netherlands)

    Lindeboom, J.J.; Mulder, B.; Vos, J.W.; Ketelaar, M.J.; Emons, A.M.C.

    2008-01-01

    Plant cell wall production is a membrane-bound process. Cell walls are composed of cellulose microfibrils, embedded inside a matrix of other polysaccharides and glycoproteins. The cell wall matrix is extruded into the existing cell wall by exocytosis. This same process also inserts the cellulose

  2. 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

  3. Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane.

    Science.gov (United States)

    Iaea, David B; Mao, Shu; Lund, Frederik W; Maxfield, Frederick R

    2017-04-15

    Cholesterol is an essential constituent of membranes in mammalian cells. The plasma membrane and the endocytic recycling compartment (ERC) are both highly enriched in cholesterol. The abundance and distribution of cholesterol among organelles are tightly controlled by a combination of mechanisms involving vesicular and nonvesicular sterol transport processes. Using the fluorescent cholesterol analogue dehydroergosterol, we examined sterol transport between the plasma membrane and the ERC using fluorescence recovery after photobleaching and a novel sterol efflux assay. We found that sterol transport between these organelles in a U2OS cell line has a t 1/2 =12-15 min. Approximately 70% of sterol transport is ATP independent and therefore is nonvesicular. Increasing cellular cholesterol levels dramatically increases bidirectional transport rate constants, but decreases in cholesterol levels have only a modest effect. A soluble sterol transport protein, STARD4, accounts for ∼25% of total sterol transport and ∼33% of nonvesicular sterol transport between the plasma membrane and ERC. This study shows that nonvesicular sterol transport mechanisms and STARD4 in particular account for a large fraction of sterol transport between the plasma membrane and the ERC. © 2017 Iaea 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).

  4. 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.

  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. 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.

  7. 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 regulator...... 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....

  8. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma

    DEFF Research Database (Denmark)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine

    2016-01-01

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic...... solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation...

  9. Spatial cycles mediated by UNC119 solubilisation maintain Src family kinases plasma membrane localisation.

    Science.gov (United States)

    Konitsiotis, Antonios D; Roßmannek, Lisaweta; Stanoev, Angel; Schmick, Malte; Bastiaens, Philippe I H

    2017-07-24

    The peripheral membrane proto-oncogene Src family protein tyrosine kinases relay growth factor signals to the cytoplasm of mammalian cells. We unravel the spatial cycles of solubilisation, trapping on perinuclear membrane compartments and vesicular transport that counter entropic equilibration to endomembranes for maintaining the enrichment and activity of Src family protein tyrosine kinases at the plasma membrane. The solubilising factor UNC119 sequesters myristoylated Src family protein tyrosine kinases from the cytoplasm, enhancing their diffusion to effectively release Src family protein tyrosine kinases on the recycling endosome by localised Arl2/3 activity. Src is then trapped on the recycling endosome via electrostatic interactions, whereas Fyn is quickly released to be kinetically trapped on the Golgi by palmitoyl acyl-transferase activity. Vesicular trafficking from these compartments restores enrichment of the Src family protein tyrosine kinases to the plasma membrane. Interference with these spatial cycles by UNC119 knockdown disrupts Src family protein tyrosine kinase localisation and signalling activity, indicating that UNC119 could be a drug target to affect oncogenic Src family protein tyrosine kinase signalling.The peripheral membrane proto-oncogene Src family protein tyrosine kinases (SFKs) transmit growth factor signals to the cytoplasm. Here the authors show that the solubilising factor UNC119 sequesters myristoylated SFKs to maintain its enrichment at the plasma membrane to enable signal transduction.

  10. 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

  11. Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages.

    Science.gov (United States)

    de la Haba, Carlos; Palacio, José R; Martínez, Paz; Morros, Antoni

    2013-02-01

    Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H(2)O(2) at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H(2)O(2) concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H(2)O(2) causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H(2)O(2), these new raft domains might be involved in cell survival pathways. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. 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

  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. 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.

  15. 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

  16. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  17. Coiled-coil oligomerization controls localization of the plasma membrane REMORINs.

    Science.gov (United States)

    Martinez, Denis; Legrand, Anthony; Gronnier, Julien; Decossas, Marion; Gouguet, Paul; Lambert, Olivier; Berbon, Mélanie; Verron, Loris; Grélard, Axelle; Germain, Veronique; Loquet, Antoine; Mongrand, Sébastien; Habenstein, Birgit

    2018-02-23

    REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo. Copyright © 2018. Published by Elsevier Inc.

  18. 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.

  19. 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.

  20. Interaction pathways between soft lipid nanodiscs and plasma membranes: A molecular modeling study.

    Science.gov (United States)

    Li, Shixin; Luo, Zhen; Xu, Yan; Ren, Hao; Deng, Li; Zhang, Xianren; Huang, Fang; Yue, Tongtao

    2017-10-01

    Lipid nanodisc, a model membrane platform originally synthesized for study of membrane proteins, has recently been used as the carrier to deliver amphiphilic drugs into target tumor cells. However, the central question of how cells interact with such emerging nanomaterials remains unclear and deserves our research for both improving the delivery efficiency and reducing the side effect. In this work, a binary lipid nanodisc is designed as the minimum model to investigate its interactions with plasma membranes by using the dissipative particle dynamics method. Three typical interaction pathways, including the membrane attachment with lipid domain exchange of nanodiscs, the partial membrane wrapping with nanodisc vesiculation, and the receptor-mediated endocytosis, are discovered. For the first pathway, the boundary normal lipids acting as ligands diffuse along the nanodisc rim to gather at the membrane interface, repelling the central bola lipids to reach a stable membrane attachment. If bola lipids are positioned at the periphery and act as ligands, they diffuse to form a large aggregate being wrapped by the membrane, leaving the normal lipids exposed on the membrane exterior by assembling into a vesicle. Finally, by setting both central normal lipids and boundary bola lipids as ligands, the receptor-mediated endocytosis occurs via both deformation and self-rotation of the nanodiscs. All above pathways for soft lipid nanodiscs are quite different from those for rigid nanoparticles, which may provide useful guidelines for design of soft lipid nanodiscs in widespread biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Measuring the Viscosity of the Escherichia coli Plasma Membrane Using Molecular Rotors.

    Science.gov (United States)

    Mika, Jacek T; Thompson, Alexander J; Dent, Michael R; Brooks, Nicholas J; Michiels, Jan; Hofkens, Johan; Kuimova, Marina K

    2016-10-04

    The viscosity is a highly important parameter within the cell membrane, affecting the diffusion of small molecules and, hence, controlling the rates of intracellular reactions. There is significant interest in the direct, quantitative assessment of membrane viscosity. Here we report the use of fluorescence lifetime imaging microscopy of the molecular rotor BODIPY C10 in the membranes of live Escherichia coli bacteria to permit direct quantification of the viscosity. Using this approach, we investigated the viscosity in live E. coli cells, spheroplasts, and liposomes made from E. coli membrane extracts. For live cells and spheroplasts, the viscosity was measured at both room temperature (23°C) and the E. coli growth temperature (37°C), while the membrane extract liposomes were studied over a range of measurement temperatures (5-40°C). At 37°C, we recorded a membrane viscosity in live E. coli cells of 950 cP, which is considerably higher than that previously observed in other live cell membranes (e.g., eukaryotic cells, membranes of Bacillus vegetative cells). Interestingly, this indicates that E. coli cells exhibit a high degree of lipid ordering within their liquid-phase plasma membranes. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. 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.

  3. 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

  4. 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...

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

    Science.gov (United States)

    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 (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. 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.

  6. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.

  7. Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

    Energy Technology Data Exchange (ETDEWEB)

    Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.; Nguyen, Amelia Y.; Gritsenko, Marina A.; Smith, Richard D.; Koppenaal, David W.; Pakrasi, Himadri B.

    2016-04-07

    Cyanobacteria are photosynthetic microbes with highlydifferentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems in cyanobacteria, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes, many more proteins remain to be identified, and a comprehensive catalogue of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 differentially localized proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared with the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared with a more specialized role for the thylakoid membrane in cellular energetics. Thus, our data clearly define the two membrane systems with distinct functions. Overall, the protein compositions of the Synechocystis 6803 plasma membrane and thylakoid membrane are quite similar to that of the plasma membrane of Escherichia coli and thylakoid membrane of Arabidopsis chloroplasts, respectively. Synechocystis 6803 can therefore be described as a Gram

  8. Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

    Science.gov (United States)

    Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  9. 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

  10. Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans.

    Czech Academy of Sciences Publication Activity Database

    Wang, H.X.; Douglas, L.M.; Veselá, Petra; Rachel, R.; Malínský, Jan; Konopka, J.B.

    2016-01-01

    Roč. 27, č. 10 (2016), s. 1663-1675 ISSN 1059-1524 R&D Projects: GA ČR(CZ) GA15-10641S Institutional support: RVO:68378041 Keywords : plasma membrane * eisosome * PIL1 * SUR7 Subject RIV: EA - Cell Biology Impact factor: 3.685, year: 2016

  11. 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...

  12. 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…

  13. 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...

  14. The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway.

    Science.gov (United States)

    Warnier, Marine; Flaman, Jean-Michel; Chouabe, Christophe; Wiel, Clotilde; Gras, Baptiste; Griveau, Audrey; Blanc, Elena; Foy, Jean-Philippe; Mathot, Pauline; Saintigny, Pierre; Van Coppenolle, Fabien; Vindrieux, David; Martin, Nadine; Bernard, David

    2018-02-15

    Oncogenic signals lead to premature senescence in normal human cells causing a proliferation arrest and the elimination of these defective cells by immune cells. Oncogene-induced senescence (OIS) prevents aberrant cell division and tumor initiation. In order to identify new regulators of OIS, we performed a loss-of-function genetic screen and identified that the loss of SCN9A allowed cells to escape from OIS. The expression of this sodium channel increased in senescent cells during OIS. This upregulation was mediated by NF-κB transcription factors, which are well-known regulators of senescence. Importantly, the induction of SCN9A by an oncogenic signal or by p53 activation led to plasma membrane depolarization, which in turn, was able to induce premature senescence. Computational and experimental analyses revealed that SCN9A and plasma membrane depolarization mediated the repression of mitotic genes through a calcium/Rb/E2F pathway to promote senescence. Taken together, our work delineates a new pathway, which involves the NF-κB transcription factor, SCN9A expression, plasma membrane depolarization, increased calcium, the Rb/E2F pathway and mitotic gene repression in the regulation of senescence. This work thus provides new insight into the involvement of ion channels and plasma membrane potential in the control of senescence. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  15. Purification of the synaptosomal plasma membrane (Ca(2+) + Mg(2+))-ATPase from pig brain.

    Science.gov (United States)

    Salvador, J M; Mata, A M

    1996-04-01

    The Ca(2+)-ATPase from the synaptosomal plasma membrane has been purified nearly to homogeneity from pig brain by a new procedure involving the calmodulin-affinity-chromatography technique. This is a convenient alternative to the standard methods for the purification of the plasma membrane Ca(2+)-ATPase from different sources that were unsuitable to purify the enzyme from pig brain. The main feature of this procedure is the use of 15% (v/v) glycerol as stabilizing agent, instead of acidic phospholipid. By using this protocol the enzyme was purified 36-fold with respect to the plasma membrane vesicle fraction, showing a specific activity of 2.3 i.u. in the presence of acidic phospholipid. In SDS/PAGE, it appears as a single protein band around Mr140 000 that can be phosphorylated by [gamma-(32)P]ATP in the presence of La(3+) and recognized by specific antibodies against the plasma membrane Ca(2+)-ATPase from pig antral smooth muscle. Calmodulin activates the enzyme 1.5-1.8-fold in the presence of phosphatidylcholine but not in the presence of phosphatidylserine.

  16. 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...

  17. Lectin receptor kinase LecRK-b2 localizes to plasma membrane and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-20

    -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 self-.

  18. 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 ...

  19. Membranes produced by plasma enhanced chemical vapor deposition technique for low temperature fuel cell applications

    Science.gov (United States)

    Ennajdaoui, Aboubakr; Roualdes, Stéphanie; Brault, Pascal; Durand, Jean

    A plasma polymerization process using a continuous glow discharge has been implemented for preparing proton conducting membranes from trifluoromethane sulfonic acid and styrene. The chemical and physical structure of plasma membranes has been investigated using FTIR and SEM. The films are homogeneous with a good adhesion on commercial gas diffusion layer (E-Tek ®). Their deposition rate can be increased with increasing flow rate and input power. The thermogravimetric analysis under air of plasma polymers has showed a thermal stability up to 140 °C. Compared to the pulsed glow discharge studied in a previous paper, the continuous glow discharge has enabled to enhance the proton conductivity of membranes by a factor 3 (up to 1.7 mS cm -1). Moreover, the low methanol permeability (methanol diffusion coefficient down to 5 × 10 -13 m 2 s -1) of membranes has been confirmed by this study. In an industrial context, a reactor prototype has been developed to manufacture by plasma processes all active layers of fuel cell cores to be integrated in original compact PEMFC or DMFC.

  20. Method of preparing water purification membranes. [polymerization of allyl amine as thin films in plasma discharge

    Science.gov (United States)

    Hollahan, J. R.; Wydeven, T. J., Jr. (Inventor)

    1974-01-01

    Allyl amine and chemically related compounds are polymerized as thin films in the presence of a plasma discharge. The monomer compound can be polymerized by itself or in the presence of an additive gas to promote polymerization and act as a carrier. The polymerized films thus produced show outstanding advantages when used as reverse osmosis membranes.

  1. 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

  2. A Hydrophobic Filter Confers the Cation Selectivity of Zygosaccharomyces rouxii Plasma-Membrane Na (+)/H (+) Antiporter

    Czech Academy of Sciences Publication Activity Database

    Kinclová-Zimmermannová, Olga; Falson, P.; Cmunt, Denis; Sychrová, Hana

    2015-01-01

    Roč. 427, č. 8 (2015), s. 1681-1697 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GAP503/10/0307; GA MŠk(CZ) LD13037 Institutional support: RVO:67985823 Keywords : yeast * plasma membrane * sodium proton exchanger * substrate specificity * potassium transport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.517, year: 2015

  3. Plasma membrane microdomains regulate turnover of transport proteins in yeast

    Czech Academy of Sciences Publication Activity Database

    Grossmann, G.; Malínský, Jan; Stahlschmidt, W.; Loibl, M.; Weig-Meckl, I.; Frommer, W.B.; Opekarová, Miroslava; Tanner, W.

    2008-01-01

    Roč. 183, č. 6 (2008), s. 1075-1088 ISSN 0021-9525 R&D Projects: GA ČR GA204/06/0009; GA ČR GA204/07/0133; GA ČR GC204/08/J024 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50200510 Keywords : Lithium acetate * Membrane compartment of Can1 * Monomeric red fluorescent protein Subject RIV: EA - Cell Biology Impact factor: 9.120, year: 2008

  4. Elevated cAMP increases aquaporin-3 plasma membrane diffusion

    DEFF Research Database (Denmark)

    Marlar, Saw; Christensen, Eva Arnspang; Koffman, Jennifer Skaarup

    2014-01-01

    Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water...... be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal...

  5. 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.

  6. Effects of intense noise exposure on the outer hair cell plasma membrane fluidity.

    Science.gov (United States)

    Chen, Guang-Di; Zhao, Hong-Bo

    2007-04-01

    Outer hair cells (OHCs) play an important role in cochlear amplification via their length changes (electromotility). A noise-induced cochlear amplification loss leading to a permanent threshold shift (PTS) was observed without a significant hair cell loss in rats [Chen, G.D., Liu, Y., 2005. Mechanisms of noise-induced hearing loss potentiation by hypoxia. Hear. Res. 200, 1-9.]. Since motor proteins are inserted in the OHC lateral membrane, any change in the OHC plasma membrane may result in a loss of OHC electromotility, leading to a loss of cochlear amplification. In this study, the lateral diffusion in the OHC plasma membrane was determined in vitro in guinea pigs by fluorescent recovery after photobleaching (FRAP) after an in vivo noise exposure. The lateral diffusion in the OHC plasma membrane demonstrated a length-dependence, which increased as OHC length increased. A reduction in the lateral diffusion was observed in those OHCs with lengths of 50-70 microm after exposure to an 8-kHz octave band noise at 110 dB SPL for 3h. This membrane fluidity change was associated with the selective PTS at frequencies around 8 kHz. The reduction of the lateral diffusion in the OHC lateral wall indicated that noise could impair the micromechanics of the OHC lateral wall and might consequently impair OHC electromotility to induce threshold shift.

  7. Model lipid bilayers mimic non-specific interactions of gold nanoparticles with macrophage plasma membranes.

    Science.gov (United States)

    Montis, Costanza; Generini, Viola; Boccalini, Giulia; Bergese, Paolo; Bani, Daniele; Berti, Debora

    2018-04-15

    Understanding the interaction between nanomaterials and biological interfaces is a key unmet goal that still hampers clinical translation of nanomedicine. Here we investigate and compare non-specific interaction of gold nanoparticles (AuNPs) with synthetic lipid and wild type macrophage membranes. A comprehensive data set was generated by systematically varying the structural and physicochemical properties of the AuNPs (size, shape, charge, surface functionalization) and of the synthetic membranes (composition, fluidity, bending properties and surface charge), which allowed to unveil the matching conditions for the interaction of the AuNPs with macrophage plasma membranes in vitro. This effort directly proved for the first time that synthetic bilayers can be set to mimic and predict with high fidelity key aspects of nanoparticle interaction with macrophage eukaryotic plasma membranes. It then allowed to model the experimental observations according to classical interface thermodynamics and in turn determine the paramount role played by non-specific contributions, primarily electrostatic, Van der Waals and bending energy, in driving nanoparticle-plasma membrane interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. 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

  9. Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

    Science.gov (United States)

    Tischner, R.; Ward, M. R.; Huffaker, R. C.

    1989-01-01

    Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

  10. 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.

  11. Glia plasma membrane transporters: Key players in glutamatergic neurotransmission.

    Science.gov (United States)

    Flores-Méndez, Marco; Mendez-Flores, Orquidia G; Ortega, Arturo

    2016-09-01

    Glutamate, the main excitatory amino acid in the central nervous system, elicits its functions through the activation of specific membrane receptors that are expressed in neurons and glial cells. The re-cycling of this amino acid is carried out mostly through a continuous interplay between neurons and glia cells, given the fact that the removal of glutamate from the synaptic cleft depends mainly on glial glutamate transporters. Therefore, a functional and physical interaction between membrane transporters links glutamate uptake, transformation to glutamine and its release to the extra-synaptic space and its uptake to the pre-synaptic terminal. This sequence of events, best known as the glutamate/glutamine shuttle is central to glutamatergic transmission. In this sense, the uptake process triggers a complex series of biochemical cascades that modify the physiology of glial cells in the immediate, short and long term so as to be capable to take up, transform and release these amino acids in a regulated amount and in an appropriate time frame to sustain glutamatergic neurotransmission. Among the signaling cascades activated in glial cells by glutamate transporters, a sustained Na(+) and Ca(2+) influx, protein posttranslational modifications and gene expression regulation at the transcriptional and translational levels are present. Therefore, it is clear that the pivotal role of glial cells in the context of excitatory transmission has been constantly underestimated. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database

    DEFF Research Database (Denmark)

    Nühse, Thomas S; Stensballe, Allan; Jensen, Ole N

    2004-01-01

    the sites. These analyses yield general principles for predicting other phosphorylation sites in plants and provide indications of specificity determinants for responsible kinases. In addition, more than 50 sites were mapped on receptor-like kinases and revealed an unexpected complexity of regulation...... database for plant phosphorylation sites maintained by PlantsP (http://plantsp.sdsc.edu) that will be updated as the project expands to encompass additional tissues and organelles....

  13. Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Fendrych, Matyáš; Synek, Lukáš; Pečenková, Tamara; Drdová, Edita; Sekereš, Juraj; De Rycke, R.; Nowack, M.K.; Žárský, Viktor

    2013-01-01

    Roč. 24, č. 4 (2013), s. 510-520 ISSN 1059-1524 R&D Projects: GA ČR(CZ) GAP305/11/1629 Institutional research plan: CEZ:AV0Z50380511 Keywords : PLANT TRANSFORMATION * POLARIZED SECRETION * MAMMALIAN EXOCYST Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.548, year: 2013

  14. 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.

  15. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    Science.gov (United States)

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  16. Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain.

    Science.gov (United States)

    Matsuura, Daisuke; Taguchi, Katsutoshi; Yagisawa, Hitoshi; Maekawa, Shohei

    2007-08-16

    Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as "lipid rafts" obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP(2) is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP(2) in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP(2) in SPM-derived DRM, we measured the amount of PIP(2) in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cdelta1 as a PIP(2) binding probe. About 10% of the PIP(2) in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP(2) in DRM prepared from SPM.

  17. 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

  18. Polyethylene glycol acrylate-grafted polysulphone membrane for artificial lungs: plasma modification and haemocompatibility improvement.

    Science.gov (United States)

    Wang, Weiping; Huang, Xin; Yin, Haiyan; Fan, Wenling; Zhang, Tao; Li, Lei; Mao, Chun

    2015-12-14

    In this study, polyethylene glycol acrylate (PEGA) was introduced onto the surface of polysulphone (PSF) membrane to prepare PSF-PEGA membranes through low-temperature plasma technology for haemocompatibility improvement of artificial lungs. The effects of plasma power, PEGA solution concentration and dipcoating temperature on surface modification were systematically investigated. Results of Fourier transform infrared spectroscopy, x-ray photoelectron spectroscopy and PEGA grafting degree confirmed that PEGA was successfully grafted onto the PSF membranes. Contact angle values showed that the hydrophilicity of the PSF-PEGA membrane surface increased by 21.5%. The results of the protein adsorption, platelet adhesion and coagulation tests further showed the excellent haemocompatibility of the modified membrane. Gas exchange tests also revealed that at a porcine blood flow rate of 5 l min(-1), O2 and CO2 exchange rates through the PSF-PEGA membrane were 198.6 and 170.9 ml min(-1), respectively; approximately this is the gas exchange capacity of commercial respiratory assistance devices.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

    by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization......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...

  20. 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.

  1. Arabidopsis thaliana Yellow Stripe1-Like4 and Yellow Stripe1-Like6 localize to internal cellular membranes and are involved in metal ion homeostasis.

    Directory of Open Access Journals (Sweden)

    Heng-Hsuan eChu

    2013-07-01

    Full Text Available Several members of the Yellow Stripe1-Like (YSL family of transporter proteins are able to transport metal-nicotianamine (NA complexes. Substantial progress has been made in understanding the roles of the Arabidopsis YSLs that are most closely related to the founding member of the family, ZmYS1 (e.g., AtYSL1, AtYSL2 and AtYSL3, but there is little information concerning members of the other two well-conserved YSL clades. Here, we provide evidence that AtYSL4 and AtYSL6, which are the only genes in Arabidopsis belong to YSL Group II, are localized to vacuole membranes and to internal membranes resembling endoplasmic reticulum. Both single and double mutants for YSL4 and YSL6 were rigorously analyzed, and have surprisingly mild phenotypes, in spite of the strong and wide-ranging expression of YSL6. However, in the presence of toxic levels of Mn and Ni, plants with mutations in YSL4 and YSL6 and plants overexpressing GFP-tagged YSL6 showed growth defects, indicating a role for these transporters in heavy metal stress responses.

  2. 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.

  3. Measuring H+ pumping and membrane potential formation in sealed membrane vesicle systems

    DEFF Research Database (Denmark)

    Wielandt, Alex Green; Palmgren, Michael Broberg; Fuglsang, Anja Thoe

    2016-01-01

    is not converted into a product and only moves a few nanometers in space. Here, we describe two methods for the measurement of active proton pumping across lipid bilayers and the concomitant formation of a membrane potential, applying the dyes 9-amino-6-chloro-2-methoxyacridine (ACMA) and oxonol VI. The methods...... are exemplified by assaying transport of the Arabidopsis thaliana plasma membrane H(+)-ATPase (proton pump), which after heterologous expression in Saccharomyces cerevisiae and subsequent purification has been reconstituted in proteoliposomes....

  4. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    Science.gov (United States)

    Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E; Fridberger, Anders; Zuo, Jian

    2015-09-01

    Nature's fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  5. Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Tetsuji Yamashita

    2015-09-01

    Full Text Available Nature's fastest motors are the cochlear outer hair cells (OHCs. These sensory cells use a membrane protein, Slc26a5 (prestin, to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5's active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.

  6. 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

  7. Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle

    2011-12-01

    Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

  8. Plasma membrane calcium channels in cancer: Alterations and consequences for cell proliferation and migration.

    Science.gov (United States)

    Déliot, Nadine; Constantin, Bruno

    2015-10-01

    The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2+ homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades involving the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a

  9. The Plasma Membrane H+-ATPase AHA1 Plays a Major Role in Stomatal Opening in Response to Blue Light.

    Science.gov (United States)

    Yamauchi, Shota; Takemiya, Atsushi; Sakamoto, Tomoaki; Kurata, Tetsuya; Tsutsumi, Toshifumi; Kinoshita, Toshinori; Shimazaki, Ken-Ichiro

    2016-08-01

    Stomata open in response to a beam of weak blue light under strong red light illumination. A blue light signal is perceived by phototropins and transmitted to the plasma membrane H(+)-ATPase that drives stomatal opening. To identify the components in this pathway, we screened for mutants impaired in blue light-dependent stomatal opening. We analyzed one such mutant, provisionally named blus2 (blue light signaling2), and found that stomatal opening in leaves was impaired by 65%, although the magnitude of red light-induced opening was not affected. Blue light-dependent stomatal opening in the epidermis and H(+) pumping in guard cell protoplasts were inhibited by 70% in blus2 Whole-genome resequencing identified a mutation in the AHA1 gene of the mutant at Gly-602. T-DNA insertion mutants of AHA1 exhibited a similar phenotype to blus2; this phenotype was complemented by the AHA1 gene. We renamed blus2 as aha1-10 T-DNA insertion mutants of AHA2 and AHA5 did not show any impairment in stomatal response, although the transcript levels of AHA2 and AHA5 were higher than those of AHA1 in wild-type guard cells. Stomata in ost2, a constitutively active AHA1 mutant, did not respond to blue light. A decreased amount of H(+)-ATPase in aha1-10 accounted for the reduced stomatal blue light responses and the decrease was likely caused by proteolysis of misfolded AHA1. From these results, we conclude that AHA1 plays a major role in blue light-dependent stomatal opening in Arabidopsis and that the mutation made the AHA1 protein unstable in guard cells. © 2016 American Society of Plant Biologists. All Rights Reserved.

  10. Evidence for a role in growth and salt resistance of a plasma membrane H+-ATPase in the root endodermis

    Science.gov (United States)

    Vitart, V.; Baxter, I.; Doerner, P.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2001-01-01

    The plasma membrane of plant cells is energized by an electrochemical gradient produced by P-type H+-ATPases (proton pumps). These pumps are encoded by at least 12 genes in Arabidopsis. Here we provide evidence that isoform AHA4 contributes to solute transport through the root endodermis. AHA4 is expressed most strongly in the root endodermis and flowers, as suggested by promoter-GUS reporter assays. A disruption of this pump (aha4-1) was identified as a T-DNA insertion in the middle of the gene (after VFP(574)). Truncated aha4-1 transcripts accumulate to approximately 50% of the level observed for AHA4 mRNA in wild-type plants. Plants homozygous for aha4-1 (-/-) show a subtle reduction in root and shoot growth compared with wild-type plants when grown under normal conditions. However, a mutant phenotype is very clear in plants grown under salt stress (e.g., 75 or 110 mM NaCl). In leaves of mutant plants subjected to Na stress, the ratio of Na to K increased 4-5-fold. Interestingly, the aha4-1 mutation appears to be semidominant and was only partially complemented by the introduction of additional wild-type copies of AHA4. These results are consistent with the hypothesis that aha4-1 may produce a dominant negative protein or RNA that partially disrupts the activity of other pumps or functions in the root endodermal tissue, thereby compromising the function of this cell layer in controlling ion homeostasis and nutrient transport.

  11. Membrane Targeting of P-type ATPases in Plant Cells

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

  12. Membrane Targeting of P-type ATPases in Plant Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey F. Harper, Ph.D.

    2004-06-30

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems.

  13. 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.

  14. Navigation through the Plasma Membrane Molecular Landscape Shapes Random Organelle Movement.

    Science.gov (United States)

    Dun, Alison R; Lord, Gabriel J; Wilson, Rhodri S; Kavanagh, Deirdre M; Cialowicz, Katarzyna I; Sugita, Shuzo; Park, Seungmee; Yang, Lei; Smyth, Annya M; Papadopulos, Andreas; Rickman, Colin; Duncan, Rory R

    2017-02-06

    Eukaryotic plasma membrane organization theory has long been controversial, in part due to a dearth of suitably high-resolution techniques to probe molecular architecture in situ and integrate information from diverse data streams [1]. Notably, clustered patterning of membrane proteins is a commonly conserved feature across diverse protein families (reviewed in [2]), including the SNAREs [3], SM proteins [4, 5], ion channels [6, 7], and receptors (e.g., [8]). Much effort has gone into analyzing the behavior of secretory organelles [9-13], and understanding the relationship between the membrane and proximal organelles [4, 5, 12, 14] is an essential goal for cell biology as broad concepts or rules may be established. Here we explore the generally accepted model that vesicles at the plasmalemma are guided by cytoskeletal tracks to specific sites on the membrane that have clustered molecular machinery for secretion [15], organized in part by the local lipid composition [16]. To increase our understanding of these fundamental processes, we integrated nanoscopy and spectroscopy of the secretory machinery with organelle tracking data in a mathematical model, iterating with knockdown cell models. We find that repeated routes followed by successive vesicles, the re-use of similar fusion sites, and the apparently distinct vesicle "pools" are all fashioned by the Brownian behavior of organelles overlaid on navigation between non-reactive secretory protein molecular depots patterned at the plasma membrane. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  15. 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.

  16. 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.

  17. Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Wojewodzka, U.; Gniadecki, R.

    2008-01-01

    -p-cyclodextrin further decreased the recovery of DiI-C-18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L-d phase lipids. (c...... containing liquid-ordered (L-o) lipids. Indeed, values of maximal fluorescence recovery after photobleaching revealed that the long-range mobility of cholera toxin B subunit (CTB, marker of L-o) was similar to 1.5-fold retarded within the focal junctions compared to the surrounding membrane. However, 1...

  18. Arabidopsis Tic40 expression in tobacco chloroplasts results in massive proliferation of the inner envelope membrane and upregulation of associated proteins.

    Science.gov (United States)

    Singh, Nameirakpam Dolendro; Li, Ming; Lee, Sueng-Bum; Schnell, Danny; Daniell, Henry

    2008-12-01

    The chloroplast inner envelope membrane (IM) plays essential roles in lipid synthesis, metabolite transport, and cellular signaling in plants. We have targeted a model nucleus-encoded IM protein from Arabidopsis thaliana, pre-Tic40-His, by relocating its expression from the nucleus to the chloroplast genome. Pre-Tic40-His was properly targeted, processed, and inserted. It attained correct topology and was folded and assembled into a TIC complex, where it accounted for up to 15% of the total chloroplast protein. These results confirm the existence of a novel pathway for protein targeting to the IM. Tic40-His overexpression resulted in a massive proliferation of the IM (up to 19 layers in electron micrographs) without significant effects on plant growth or reproduction. Consistent with IM proliferation, the expression levels of other endogenous IM proteins (IEP37, PPT, Tic110) were significantly (10-fold) upregulated but those of outer envelope membrane (Toc159), stromal (hsp93, cpn60), or thylakoid (LHCP, OE23) proteins were not increased, suggesting retrograde signal transduction between chloroplast and nuclear genomes to increase lipid and protein components for accommodation of increased accumulation of Tic40. This study opens the door for understanding the regulation of membrane biogenesis within the organelle and the utilization of transgenic chloroplasts as bioreactors for hyperaccumulation of membrane proteins for biotechnological applications.

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

    Science.gov (United States)

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

    2016-02-01

    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 than 99.99%. This is the first time that an investigation demonstrates how the permeation characteristics of these membranes is directly related to data from spectral, morphological and surface charge analyses, which provide new insights on the impact of plasma treatments on both, the surface charge and roughness, of PTFE porous materials.

  20. Effect of PAF on polyrnorphonuclear leucocyte plasma membrane polarity: a fluorescence study

    Directory of Open Access Journals (Sweden)

    A. Kantar

    1993-01-01

    Full Text Available The effect of PAF on the plasma membrane polarity of polymorphonuclear leukocytes (PMNs was investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-1auroyl naphthalene (Laurdan, which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surrounding. Laurdan shows a marked steady-state emission blue-shift in non-polar solvents, with respect to polar solvents. Our results demonstrate that PAF (10−7 M induces a blue shift of the fluorescence emission spectra of Laurdan. These changes are blocked in the presence of the PAF antagonist, L-659,989. Our data indicate that the interaction between PAF and PMNs is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.

  1. Plasma membrane reorganization induced by tumor promoters in an epithelial cell line

    Energy Technology Data Exchange (ETDEWEB)

    PACKARD, BEVERLY S.; SAXTON, MICHAEL J.; BISSELL, MINA J.; KLEIN, MELVIN P.

    1984-01-01

    The effects of phorbol ester tumor promoters on the lateral diffusion in plasma membrane lipid environments were examined by the technique of fluorescence recovery after photobleaching. To this end, the probe collarein, a fluorescent lipid analog that has the property of exclusive localization in the plasma membrane, was synthesized. Measured decreases in three parameters [percentage of fluorescence bleached (30%), percentage of recovery (52%), and half-time for recovery (52%)] connoted the appearance of an immobile fraction upon exposure to tumor promoters. These data are consistent with lipid reorganization in response to a reorganization of the intra- and perimembranous macromolecular scaffolding upon the interaction of cells with tumor promoters. The idea of induced reorganization is supported by experiments in which cell shape change, brought about by either exposure to cytochalasin B or growth on matrices of collagen, fibronectin, or laminin, resulted in values in the fluorescence recovery after photobleaching technique similar to those with active phorbol esters.

  2. Cell-geometry-dependent changes in plasma membrane order direct stem cell signalling and fate.

    Science.gov (United States)

    von Erlach, Thomas C; Bertazzo, Sergio; Wozniak, Michele A; Horejs, Christine-Maria; Maynard, Stephanie A; Attwood, Simon; Robinson, Benjamin K; Autefage, Hélène; Kallepitis, Charalambos; Del Río Hernández, Armando; Chen, Christopher S; Goldoni, Silvia; Stevens, Molly M

    2018-03-01

    Cell size and shape affect cellular processes such as cell survival, growth and differentiation 1-4 , thus establishing cell geometry as a fundamental regulator of cell physiology. The contributions of the cytoskeleton, specifically actomyosin tension, to these effects have been described, but the exact biophysical mechanisms that translate changes in cell geometry to changes in cell behaviour remain mostly unresolved. Using a variety of innovative materials techniques, we demonstrate that the nanostructure and lipid assembly within the cell plasma membrane are regulated by cell geometry in a ligand-independent manner. These biophysical changes trigger signalling events involving the serine/threonine kinase Akt/protein kinase B (PKB) that direct cell-geometry-dependent mesenchymal stem cell differentiation. Our study defines a central regulatory role by plasma membrane ordered lipid raft microdomains in modulating stem cell differentiation with potential translational applications.

  3. Surface functionalization dependent subcellular localization of Superparamagnetic nanoparticle in plasma membrane and endosome.

    Science.gov (United States)

    Thimiri Govinda Raj, Deepak B; Khan, Niamat Ali

    2018-01-01

    In this article, we elaborate the application of thermal decomposition based synthesis of Fe 3 O 4 superparamagnetic nanoparticle (SPMNP) in subcellular fractionation context. Here, we performed surface functionalization of SPMNP with phospholipids and dimercaptosuccinic acid. Surprisingly, we observed surface functionalization dependent SPMNP localization in subcellular compartments such as plasma membrane, endosomes and lysosomes. By using SPMNP based subcellular localization with pulse-chase methodology, we could use SPMNP for high pure-high yield organelle (plasma membrane, endosomes and lysosome) fractionation. Further, SPMNP that are distinctly localized in subcellular compartments can be used as technology for subcellular fractionation that can complement existing tools for cell biology research. As a future perspective, isolated magnetic organelles can be extended to protein/protein complex purification for biochemical and structural biology studies.

  4. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  5. Proliferation and differentiation of stem cells in contact with eluate from fibrin-rich plasma membrane.

    Science.gov (United States)

    Souza, Fernanda Gimenez de; Fernandes, Beatriz Luci; Rebelatto, Carmen Lucia Kuniyoshi; Aguiar, Alessandra Melo de; Fracaro, Letícia; Brofman, Paulo Roberto Slud

    2018-01-01

    To evaluate the ability of the eluate from fibrin-rich plasma (FRP) membrane to induce proliferation and differentiation of isolated human adipose-derived stem cells (ASCs) into chondrocytes. FRP membranes were obtained by centrifugation of peripheral blood from two healthy donors, cut, and maintained in culture plate wells for 48 h to prepare the fibrin eluate. The SCATh were isolated from adipose tissue by collagenase digestion solution, and expanded in vitro . Cells were expanded and treated with DMEM-F12 culture, a commercial media for chondrogenic differentiation, and eluate from FRP membrane for three days, and labeled with BrdU for quantitative assessment of cell proliferation using the High-Content Operetta ® imaging system. For the chondrogenic differentiation assay, the SCATh were grown in micromass for 21 days and stained with toluidine blue and aggrecan for qualitative evaluation by light microscopy. The statistical analysis was performed using ANOVA and Tukey's test. There was a greater proliferation of cells treated with the eluate from FRP membrane compared to the other two treatments, where the ANOVA test showed significance ( p  membrane stimulated cell proliferation and induced differentiation of the stem cells into chondrocytes, suggesting a potential application of FRP membranes in hyaline cartilage regeneration therapies.

  6. Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis.

    Science.gov (United States)

    Sameni, Sara; Malacrida, Leonel; Tan, Zhiqun; Digman, Michelle A

    2018-01-15

    Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

  7. Myelin basic protein induces neuron-specific toxicity by directly damaging the neuronal plasma membrane.

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    Full Text Available The central nervous system (CNS insults may cause massive demyelination and lead to the release of myelin-associated proteins including its major component myelin basic protein (MBP. MBP is reported to induce glial activation but its effect on neurons is still little known. Here we found that MBP specifically bound to the extracellular surface of the neuronal plasma membrane and induced neurotoxicity in vitro. This effect of MBP on neurons was basicity-dependent because the binding was blocked by acidic lipids and competed by other basic proteins. Further studies revealed that MBP induced damage to neuronal membrane integrity and function by depolarizing the resting membrane potential, increasing the permeability to cations and other molecules, and decreasing the membrane fluidity. At last, artificial liposome vesicle assay showed that MBP directly disturbed acidic lipid bilayer and resulted in increased membrane permeability. These results revealed that MBP induces neurotoxicity through its direct interaction with acidic components on the extracellular surface of neuronal membrane, which may suggest a possible contribution of MBP to the pathogenesis in the CNS disorders with myelin damage.

  8. 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

  9. Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

    Science.gov (United States)

    Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

    2018-02-28

    Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

  10. 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.

  11. Silymarin protects plasma membrane and acrosome integrity in sperm treated with sodium arsenite

    Directory of Open Access Journals (Sweden)

    Farzaneh Eskandari

    2016-01-01

    Full Text Available Background: Exposure to arsenic is associated with impairment of male reproductive function by inducing oxidative stress. Silymarin with an antioxidant property scavenges free radicals. Objective: The aim of this study was to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm plasma membrane and acrosome integrity. Materials and Methods: Ram epidydimal spermatozoa were divided into five groups: spermatozoa at 0 hr, spermatozoa at 180 min (control, spermatozoa treated with silymarin (20 μM + sodium arsenite (10 μM for 180 min, spermatozoa treated with sodium arsenite (10 μM for 180 min and spermatozoa treated with silymarin (20 μM for 180 min. Double staining of Hoechst and propidium iodide was performed to evaluate sperm plasma membrane integrity, whereas comassie brilliant blue staining was used to assess acrosome integrity. Results: Plasma membrane (p< 0.001 and acrosome integrity (p< 0.05 of the spermatozoa were significantly reduced in sodium arsenite group compared to the control. In silymarin + sodium arsenite group, silymarin was able to significantly (p< 0.001 ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group. The incubation of sperm for 180 min (control group showed a significant (p< 0.001 decrease in acrosome integrity compared to the spermatozoa at 0 hour. The application of silymarin alone for 180 min could also significantly (p< 0.05 increase sperm acrosome integrity compared to the control. Conclusion: Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on the ram sperm plasma membrane and acrosome integrity.

  12. Origin and development of plasma membrane derived invaginations in Vinca rosea l.

    Science.gov (United States)

    Mahlberg, P.; Walkinshaw, C.; Olson, K.

    1971-01-01

    The occurrence, morphology, and possible ontogeny of plasma-membrane-related structures are described which can develop into invaginations or intravacuolar formations. An underlying study of meristematic tissues from the shoot of Vinca rosea supports the interpretation that endocytosis does occur in plant cells and that it is appropriate to refer to these structures as endocytoses. The function of these invaginations or their content remains to be elucidated.

  13. Biotransformation of endorphins by a synaptosomal plasma membrane preparation of rat brain and by human serum

    NARCIS (Netherlands)

    Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de; Wied, D. de

    1979-01-01

    β-Endorphin (β-LPH 61–91), γ-endorphin (61–77), des-tyrosine-γ-endorphin (62–77), α-endorphin (61–76), and β-LPH 61–69 either labeled with [125I] at the N-terminal 61-tyrosine residue or unlabeled were incubated with a crude synaptosomal plasma membrane fraction of rat brain or in human serum. At

  14. Plasma surface modification of chitosan membranes : characterization and preliminary cell response studies

    OpenAIRE

    Silva, Simone Santos; Luna, Sandra M.; Gomes, Manuela E.; Benesch, Johan; Pashkuleva, I.; Mano, J. F.; Reis, R. L.

    2008-01-01

    Surface modification of biomaterials is a way to tailor cell responses whilst retaining the bulk properties. In this work, chitosan membranes were prepared by solvent casting and treated with nitrogen or argon plasma at 20Wfor 10–40 min. AFM indicated an increase in the surface roughness as a result of the ongoing etching process. XPS and contact angle measurements showed different surface elemental compositions and higher surface free energy. The MTS test and direct contact...

  15. Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells.

    Science.gov (United States)

    Chtcheglova, Lilia A; Hinterdorfer, Peter

    2018-01-01

    Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. 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.

  17. Plasma membrane is the site of productive HIV-1 particle assembly.

    Directory of Open Access Journals (Sweden)

    Nolwenn Jouvenet

    2006-12-01

    Full Text Available Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

  18. Autophagy modulates dynamics of connexins at the plasma membrane in a ubiquitin-dependent manner

    Science.gov (United States)

    Bejarano, Eloy; Girao, Henrique; Yuste, Andrea; Patel, Bindi; Marques, Carla; Spray, David C.; Pereira, Paulo; Cuervo, Ana Maria

    2012-01-01

    Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins. PMID:22496425

  19. Identification of chitosan oligosaccharides binding proteins from the plasma membrane of wheat leaf cell.

    Science.gov (United States)

    Liu, Dongdong; Jiao, Siming; Cheng, Gong; Li, Xueming; Pei, Zhichao; Pei, Yuxin; Yin, Heng; Du, Yuguang

    2018-01-26

    Chitosan oligosaccharides (COS) have the ability to improve plant resistance to pests and diseases through activating plant immune system. However, it remains unclear whether stimulating reason of COS was associated with the plasma membrane proteins. Here, the interaction of COS with wheat leaf cell demonstrated that fluorescence-labeled COS were enriched on the cell surface and the interaction of COS with plasma membrane proteins was confirmed by quartz crystal microbalance (QCM) biosensor. What's more, HPLC and SDS-PAGE analysis showed that COS binding proteins exhibited more than three peaks and the molecular weight were 66 kDa to 97 kDa, where the COS binding proteins were fished out from wheat plasma membrane proteins by the COS affinity column. More importantly, LC-MS/MS analysis demonstrated that several candidates, including W5G2U8_WHEAT (a potential wall-associated receptor kinase protein), W5HY42_WHEAT and W5I0R4_WHEAT (potential G-type lectin S-receptor-like serine/threonine-protein kinase), have the potential to be COS receptors. Copyright © 2018. Published by Elsevier B.V.

  20. Alteration of plasma membrane-bound redox systems of iron deficient pea roots by chitosan.

    Science.gov (United States)

    Meisrimler, Claudia-Nicole; Planchon, Sebastien; Renaut, Jenny; Sergeant, Kjell; Lüthje, Sabine

    2011-08-12

    Iron is essential for all living organisms and plays a crucial role in pathogenicity. This study presents the first proteome analysis of plasma membranes isolated from pea roots. Protein profiles of four different samples (+Fe, +Fe/Chitosan, -Fe, and -Fe/Chitosan) were compared by native IEF-PAGE combined with in-gel activity stains and DIGE. Using DIGE, 89 proteins of interest were detected in plasma membrane fractions. Data revealed a differential abundance of several spots in all samples investigated. In comparison to the control and -FeCh the abundance of six protein spots increased whereas 56 spots decreased in +FeCh. Altered protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. Besides stress-related proteins, transport proteins and redox enzymes were identified. Activity stains after native PAGE and spectrophotometric measurements demonstrated induction of a ferric-chelate reductase (-Fe) and a putative respiratory burst oxidase homolog (-FeCh). However, the activity of the ferric-chelate reductase decreased in -Fe plants after elicitor treatment. The activity of plasma membrane-bound class III peroxidases increased after elicitor treatment and decreased under iron-deficiency, whereas activity of quinone reductases decreased mostly after elicitor treatment. Possible functions of proteins identified and reasons for a weakened pathogen response of iron-deficient plants were discussed. Copyright © 2011 Elsevier B.V. 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. [Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

    Science.gov (United States)

    Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

    2012-01-01

    The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

  3. Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content.

    Science.gov (United States)

    Bali, Rachna; Savino, Laura; Ramirez, Diego A; Tsvetkova, Nelly M; Bagatolli, Luis; Tablin, Fern; Crowe, John H; Leidy, Chad

    2009-06-01

    There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 degrees C, but not at 37 degrees C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets ( approximately 15 mol%), appears to be crucial for the formation of large domains during cooling.

  4. One-step extraction of polar drugs from plasma by parallel artificial liquid membrane extraction.

    Science.gov (United States)

    Pilařová, Veronika; Sultani, Mumtaz; Ask, Kristine Skoglund; Nováková, Lucie; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2017-02-01

    The new microextraction technique named parallel artificial liquid membrane extraction (PALME) was introduced as an alternative approach to liquid-liquid extraction of charged analytes from aqueous samples. The concept is based on extraction of analytes across a supported liquid membrane sustained in the pores of a thin polymeric membrane, a well-known extraction principle also used in hollow fiber liquid-phase microextraction (HF-LPME). However, the new PALME technique offers a more user-friendly setup in which the supported liquid membrane is incorporated in a 96 well plate system. Thus, high-throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample preparation is performed in one step. With optimized conditions, the extraction recoveries were in the range 50-89% from human plasma after 45min extraction. The data from the method evaluation were satisfactory and in line with current guidelines, and revealed an extraction method with substantial potential for high throughput bioanalysis of polar basic drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. 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

    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...... (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...... 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...

  6. 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...

  7. 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....

  8. 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.

  9. Reduction in lateral lipid mobility of lipid bilayer membrane by atmospheric pressure plasma irradiation

    Science.gov (United States)

    Suda, Yoshiyuki; Tero, Ryugo; Yamashita, Ryuma; Yusa, Kota; Takikawa, Hirofumi

    2016-03-01

    Plasma medicine is an emerging research field in which various applications of electrical discharge, especially in the form of nonequilibrium plasma at atmospheric pressure, are examined, for example, the application of plasma to biological targets for various purposes such as selective killing of tumor cells and blood stanching. We have focused on the behavior of an artificial cell membrane system at the solid-liquid interface. To evaluate the lateral lipid mobility, we measured the diffusion coefficient of the supported lipid bilayer (SLB) composed of dioleoylphosphatidylcholine with fluorescence recovery after photobleaching by confocal laser scanning microscopy. It was found that the diffusion coefficient was decreased by plasma irradiation and that the diffusion coefficient decreasing rate proceeded with increasing plasma power. We investigated the effects of stimulation with an equilibrium chemical, H2O2, on the SLB and confirmed that the diffusion coefficient did not change at least up to a H2O2 concentration of 5 mM. These results indicate that transient active species generated by plasma play critical roles in the reduction in SLB fluidity. The effects of the two generated major oxidized lipid species, hydroxyl- or hydroperoxy-phosphatidylcholine (PC) and acyl-chain-truncated PCs terminated with aldehyde or carboxyl group, on lateral lipid mobility are discussed.

  10. The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

    DEFF Research Database (Denmark)

    Bojsen, Rasmus; Torbensen, Rasmus; Larsen, Camilla Eggert

    2013-01-01

    that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma...... observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids....

  11. Two stage binding of glucagon to receptors in rat liver plasma membranes

    International Nuclear Information System (INIS)

    Wyborski, R.J.; Horwitz, E.M.; Gurd, R.S.

    1986-01-01

    A homogeneous class of noncooperative receptors in isolated rat hepatocytes undergoes a time- and temperature-dependent conformation change with glucagon binding. A comparable system exists in rat liver plasma membranes. Dissociation assays (30 0 C) quantify the number of receptors in each conformational state. Membranes incubated without GTP demonstrated two dissociation rates. The fraction of hormone bound to the high affinity state increases with incubation time to a limiting value. With isolated membranes and a concentration of 0.2 nM [( 125 I)Iodotyrosyl 10 ]glucagon, the fraction of the high affinity form is significantly greater than that found in isolated hepatocytes. Previous work without GTP indicated that a lack of cooperativity characterized the liver membrane system. Incubation of membranes with 0.1 mM GTP increases the K/sub D/ as determined by competition assays while the slope factor (.98 +/- 0.04) indicated noncooperativity. Furthermore, in the presence of GTP a significantly greater proportion of receptors is in the low affinity state while in the absence of GTP more are in the high affinity state. The data are consistent with a mechanism by which GTP diminishes the conversion of the low affinity state to the high affinity state

  12. Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism.

    Science.gov (United States)

    Liu, Dongmin; Ren, Min; Bing, Xinyu; Stotts, Corey; Deorah, Sundeep; Love-Homan, Laurie; Dillon, Joseph S

    2006-08-01

    Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.

  13. Practical techniques for bovine sperm simultaneous fluorimetric assessment of plasma, acrosomal and mitochondrial membranes.

    Science.gov (United States)

    Celeghini, E C C; de Arruda, R P; de Andrade, A F C; Nascimento, J; Raphael, C F

    2007-10-01

    This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.

  14. Aqueous two-phase partition applied to the isolation of plasma membranes and Golgi apparatus from cultured mammalian cells

    Science.gov (United States)

    Morre, D. M.; Morre, D. J.

    2000-01-01

    Partitioning in dextran-poly(ethylene)glycol (PEG) aqueous-aqueous phase systems represents a mature technology with many applications to separations of cells and to the preparation of membranes from mammalian cells. Most applications to membrane isolation and purification have focused on plasma membranes, plasma membrane domains and separation of right side-out and inside-out plasma membrane vesicles. The method exploits a combination of membrane properties, including charge and hydrophobicity. Purification is based upon differential distributions of the constituents in a sample between the two principal compartments of the two phases (upper and lower) and at the interface. The order of affinity of animal cell membranes for the upper phase is: endoplasmic reticulum Salt concentrations and temperature affect partitioning behavior and must be precisely standardized. In some cases, it is more fortuitous to combine aqueous two-phase partition with other procedures to obtain a more highly purified preparation. A procedure is described for preparation of Golgi apparatus from transformed mammalian cells that combines aqueous two-phase partition and centrifugation. Also described is a periodic NADH oxidase, a new enzyme marker for right side-out plasma membrane vesicles not requiring detergent disruptions for measurement of activity.

  15. The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

    Science.gov (United States)

    Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

  16. 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...

  17. Loss of membrane fluidity and endocytosis inhibition are involved in rapid aluminum-induced root growth cessation in Arabidopsis thaliana

    Czech Academy of Sciences Publication Activity Database

    Krtková, J.; Havelková, L.; Křepelová, A.; Fišer, R.; Vosolsobě, S.; Novotná, Z.; Martinec, Jan; Schwarzerová, K.

    2012-01-01

    Roč. 60, Nov 2012 (2012), s. 88-97 ISSN 0981-9428 R&D Projects: GA ČR GA522/05/0340 Grant - others:GA ČR(CZ) GPP207/12/P890 Program:GP Institutional research plan: CEZ:AV0Z50380511 Keywords : Aluminum toxicity * Arabidopsis thaliana * Cortical microtubules Subject RIV: ED - Physiology Impact factor: 2.775, year: 2012

  18. Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

    Science.gov (United States)

    Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

    2013-04-01

    Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013

  19. High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes.

    Science.gov (United States)

    Malaguti, Marco; Baldini, Marta; Angeloni, Cristina; Biagi, Pierluigi; Hrelia, Silvana

    2008-06-01

    The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fatty-acid (PUFA) -supplemented diet on anthropometric parameters, erythrocyte-membrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n = 5) followed the Mediterranean diet, and the other (n = 6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation.A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice.

  20. Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections.

    Science.gov (United States)

    Koh, Serry; André, Aurélie; Edwards, Herb; Ehrhardt, David; Somerville, Shauna

    2005-11-01

    Subcellular events of Erysiphe cichoracearum infections of epidermal cells were visualized in living tissues of Arabidopsis plants carrying various green fluorescent protein (GFP)-tagged organelles via laser scanning confocal microscopy. Early in the infection sequence, cytoplasm and organelles moved towards penetration sites and accumulated near penetration pegs. Peroxisomes appeared to accumulate preferentially relative to the cytoplasm at penetration sites. Another early event, which preceded haustorium formation, was the aggregation of some GFP-tagged plasma membrane marker proteins into rings around penetration sites, which extended across cell-wall boundaries into neighboring cells. This feature localized to sites where papillae were deposited. The extrahaustorial membrane (EHM) encases the fungal feeding structure, the haustorium, separating it from the host cytoplasm. Eight plasma membrane markers were excluded from the EHM and remained in a collar-like formation around the haustorial neck. These observations support the suggestions that the EHM is a unique, specialized membrane and is different from the plasma membrane. Our results suggested two possibilities for the origin of the EHM: invagination of the plasma membrane coupled with membrane differentiation; or de novo synthesis of the EHM by targeted vesicle trafficking.

  1. XopN-T3SS effector of Xanthomonas axonopodis pv. punicae localizes to the plasma membrane and modulates ROS accumulation events during blight pathogenesis in pomegranate.

    Science.gov (United States)

    Kumar, Rishikesh; Soni, Madhvi; Mondal, Kalyan K

    2016-12-01

    Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease of pomegranate. Xap secretes effector proteins via type III secretion system (T3SS) to suppress pathogen-associated molecular pattern (PAMP)-triggered plant immunity (PTI). Previously we reported that XopN, a conserved effector of Xap, modulate in planta bacterial growth, and blight disease. In continuation to that here we report the deletion of XopN from Xap caused higher accumulation of reactive oxygen species (ROS) including H 2 O 2 and O 2 - . We quantitatively assessed the higher accumulation of H 2 O 2 in pomegranate leaves infiltrated with Xap ΔxopN compared to Xap wild-type. We analysed that 1.5 to 3.3 fold increase in transcript expression of ROS and flg22-inducible genes, namely FRK1, GST1, WRKY29, PR1, PR2 and PR5 in Arabidopsis when challenged with Xap ΔxopN; contrary, the up-regulation of all the genes were compromised when challenged with either Xap wild-type or Xap ΔxopN+xopN. Further, we demonstrated the plasma-membrane based localization of XopN protein both in its natural and experimental hosts. All together, the present study suggested that XopN-T3SS effector of Xap gets localized in the plasma membrane and suppresses ROS-mediated early defense responses during blight pathogenesis in pomegranate. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. 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.

  3. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    Directory of Open Access Journals (Sweden)

    Elena Matteucci

    2007-01-01

    Full Text Available Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs are transmembrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

  4. 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

  5. Insulin stimulates the tyrosine phosphorylation of a Mr = 160,000 glycoprotein in adipocyte plasma membranes

    International Nuclear Information System (INIS)

    Yu, K.T.; Khalaf, N.; Czech, M.P.

    1986-01-01

    In an attempt to identify putative substrates for the insulin receptor kinase, adipocyte plasma membranes were incubated with [γ- 32 P]ATP in the presence and absence of insulin. Insulin stimulates the tyrosine phosphorylation of its receptor β subunit but does not detectably alter the phosphorylation of other membrane proteins. In contrast, when plasma membranes from insulin-treated adipocytes are phosphorylated, the 32 P-labeling of a Mr=160,000 species (p160) and insulin receptor β subunit are markedly increased when compared to controls. p160 exhibits a rapid response (max. at 1 min) and high sensitivity (ED 50 = 2 x 10 -10 M) to insulin. The stimulatory effect of insulin on the phosphorylation of p160 is rapidly reversed following the addition of anti-insulin serum. Cold chase experiments indicate that insulin promotes the phosphorylation of p160 rather than inhibiting its dephosphorylation. p160 is a glycoprotein as evidenced by its adsorption to immobilized lectins and does not represent the insulin receptor precursor. The action of insulin on p160 tyrosine phosphorylation is mimicked by concanavalin A but not by EGF and other insulin-like agents such as hydrogen peroxide and vanadate. These results suggest that p160 tyrosine phosphorylation is an insulin receptor-mediated event and may participate in signalling by the insulin receptor

  6. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

    Science.gov (United States)

    Spalding, E. P.; Cosgrove, D. J.

    1992-01-01

    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  7. DC-SIGN and Influenza Hemagglutinin Dynamics in Plasma Membrane Microdomains Are Markedly Different

    Science.gov (United States)

    Itano, Michelle S.; Neumann, Aaron K.; Liu, Ping; Zhang, Feng; Gratton, Enrico; Parak, Wolfgang J.; Thompson, Nancy L.; Jacobson, Ken

    2011-01-01

    DC-SIGN, a Ca2+-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis. PMID:21641311

  8. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Osamu Sano

    Full Text Available ATP-binding cassette A1 (ABCA1, ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

  9. Influence of phospholipid environment on the phosphatidylethanolamine: ceramide-phosphorylethanolamine transferase activity in rat liver plasma membranes.

    Science.gov (United States)

    Nikolova, M N; Petkova, D H; Koumanov, K S

    1992-03-01

    1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment.

  10. Hsp70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages.

    Science.gov (United States)

    Vega, Virginia L; Rodríguez-Silva, Monica; Frey, Tiffany; Gehrmann, Mathias; Diaz, Juan Carlos; Steinem, Claudia; Multhoff, Gabriele; Arispe, Nelson; De Maio, Antonio

    2008-03-15

    Heat shock proteins (hsps) are intracellular chaperones that play a key role in the recovery from stress. Hsp70, the major stress-induced hsp, has been found in the extracellular medium and is capable of activating immune cells. The mechanism involved in Hsp70 release is controversial because this protein does not present a consensual secretory signal. In this study, we have shown that Hsp70 integrates into artificial lipid bilayer openings of ion conductance pathways. In addition, this protein was found inserted into the plasma membrane of cells after stress. Hsp70 was released into the extracellular environment in a membrane-associated form, sharing the characteristics of this protein in the plasma membrane. Extracellular membranes containing Hsp70 were at least 260-fold more effective than free recombinant protein in inducing TNF-alpha production as an indicator of macrophage activation. These observations suggest that Hsp70 translocates into the plasma membrane after stress and is released within membranous structures from intact cells, which could act as a danger signal to activate the immune system.

  11. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    Energy Technology Data Exchange (ETDEWEB)

    Backues, Steven K. [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States); Bednarek, Sebastian Y., E-mail: sybednar@wisc.edu [Department of Biochemistry, University of Wisconsin - Madison, 433 Babcock Dr., Madison, WI 53706 (United States)

    2010-03-19

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  12. Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

    International Nuclear Information System (INIS)

    Backues, Steven K.; Bednarek, Sebastian Y.

    2010-01-01

    The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion and do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.

  13. The Arabidopsis NPF3 protein is a GA transporter

    DEFF Research Database (Denmark)

    Tal, Iris; Zhang, Yi; Jørgensen, Morten Egevang

    2016-01-01

    Gibberellins (GAs) are plant hormones that promote a wide range of developmental processes. While GA signalling is well understood, little is known about how GA is transported or how GA distribution is regulated. Here we utilize fluorescently labelled GAs (GA-Fl) to screen for Arabidopsis mutants...... deficient in GA transport. We show that the NPF3 transporter efficiently transports GA across cell membranes in vitro and GA-Fl in vivo. NPF3 is expressed in root endodermis and repressed by GA. NPF3 is targeted to the plasma membrane and subject to rapid BFA-dependent recycling. We show that abscisic acid...... (ABA), an antagonist of GA, is also transported by NPF3 in vitro. ABA promotes NPF3 expression and GA-Fl uptake in plants. On the basis of these results, we propose that GA distribution and activity in Arabidopsis is partly regulated by NPF3 acting as an influx carrier and that GA-ABA interaction may...

  14. Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

    Science.gov (United States)

    Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

    2014-03-01

    Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

  15. Cholesterol Oxidase/Triton X-100 Parked Microelectrodes for the Detection of Cholesterol in Plasma Membrane at Single Cells.

    Science.gov (United States)

    Xu, Haiyan; Zhou, Shuai; Jiang, Dechen; Chen, Hong-Yuan

    2018-01-16

    The classic electrochemical analysis of plasma membrane cholesterol at single cells utilizes a cholesterol oxidase modified microelectrode that oxidizes local cholesterol efflux from the plasma membrane to generate hydrogen peroxide for the electrochemical quantification. In this letter, a mixture of cholesterol oxidase and Triton X-100 was filled in the microcapillary that could park at the Pt layer coated tip due to slow hydrodynamic flow. During the contact of the tip with the cellular membrane, Triton X-100 at the tip permeabilized the contacted membrane to release cholesterol for the reaction with cholesterol oxidase. As compared with the linkage of cholesterol oxidase at the electrode surface, the oxidase parked in aqueous solution at the tip had a higher turnover rate resulting in larger electrochemical signal for single cell analysis. More charge collected at acyl-coA:cholesterol acyltransferase (ACAT) inhibited cells supported that this novel detection strategy could monitor the flunctation of membrane cholesterol at single cells. The successful detection of plasma membrane cholesterol at single cells using the oxidase parked microelectrode will provide a special strategy for the fabrication of biosensor that permits the integration of more molecules without functional groups at the electrode to measure active and inactive molecules in the plasma membrane. Moreover, the larger electrochemical signals collected could further increase the spatial resolution for single cell electrochemical analysis.

  16. 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.

  17. The effect of propofol on plasma membrane ultrastructure in the intact cells

    Science.gov (United States)

    Jin, Weixiang; Pralle, Arnd

    The mechanism of general anesthesia is still unknown. One drug used for human anesthesia, propofol, has been shown to interact with some ligand gated ion-channels, but also easily dissolves in the lipid bilayer and alters fluidity. Which mechanism dominates or even how anesthesia arises are unclear. We study the influence of propofol on plasma membrane (PM) ultrastructure in intact cells. In the PM, transient submicroscopic nanodomains form by interactions between lipid-acyl-chains or lipid head groups, stabilized by cholesterol. In addition, membrane cytoskeleton further regulates the nanodomains, which then regulate signaling. We study transient propofol effects on these domains from low to clinically relevant propofol concentrations by analyzing diffusion of GFP-tagged outer leaflet/inner leaflet membrane proteins. Using bimFCS we measure diffusion on multiple length scales simultaneously. We observe that at low propofol concentrations, the nanodomains trap GPI-mGFP less, consistent with studies showing that propofol decreases the phase transition temperature of membrane derived vesicles. Interestingly, at clinical relevant concentrations of propofol, the nanodomains trap GPI-mGFP more strongly. This is only observed at 37C. By inhibiting myosin activity or actin filaments (de-)polymerization, we find that the activity of actin filaments further alters the behavior of cholesterol nanodomains due to propofol. We compare the effect of propofol and its analog confirming specificity.

  18. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

    Science.gov (United States)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

    2017-10-01

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  19. Intact transmembrane isoforms of the neural cell adhesion molecule are released from the plasma membrane

    DEFF Research Database (Denmark)

    Olsen, M; Krog, L; Edvardsen, K

    1993-01-01

    . By density-gradient centrifugation it was shown that shed transmembrane NCAM-B was present in fractions of high, as well as low, density, indicating that a fraction of the shed NCAM is associated with minor plasma membrane fragments. Finally, it was shown that isolated soluble NCAM inhibited cell binding......-s1 and NCAM-s2 and the function of soluble NCAM forms were investigated. It was shown that all three soluble forms could be released from brain membranes with M(r) values identical to the three major membrane-associated forms: the large transmembrane 190,000-M(r) form (NCAM-A), the smaller...... intact soluble form from membranes of cells transfected with this isoform. Thus, NCAM-s1 and NCAM-s2 probably represent intact released transmembrane NCAM-A and NCAM-B. The soluble transmembrane forms are likely to exist in vivo, as NCAM-s1 and NCAM-s2 were readily demonstrated in cerebrospinal fluid...

  20. One-step extraction of polar drugs from plasma by Parallel Artificial Liquid Membrane Extraction

    DEFF Research Database (Denmark)

    Pilařová, Veronika; Sultani, Mumtaz; Ask, Kristine Skoglund

    2017-01-01

    -throughput is achievable, in addition to the green chemistry offered by using PALME. The consumption of organic solvent is minimized to 3-5μL per sample. With a sample volume of 250μL and acceptor solution volume of 50μL, a maximal enrichment factor of five is achievable. Based on these parameters, a new method...... for extraction of polar basic drugs was developed in the present work. The basic drugs hydralazine, ephedrine, metaraminol, salbutamol, and cimetidine were used as model analytes, and were extracted from alkalized human plasma into an aqueous solution via the supported liquid membrane. The extraction...... was promoted by a carrier dissolved in the membrane, creating a temporary ion-pair complex between the hydrophilic drug and the carrier. As the model analytes were extracted directly into an aqueous solution, there was no need for evaporation of the extract before injection into LC-MS. Hence, the sample...

  1. Visualization of phosphatidic acid fluctuations in the plasma membrane of living cells.

    Directory of Open Access Journals (Sweden)

    José P Ferraz-Nogueira

    Full Text Available We developed genetically-encoded fluorescent sensors based on Förster Resonance Energy Transfer to monitor phosphatidic acid (PA fluctuations in the plasma membrane using Spo20 as PA-binding motif. Basal PA levels and phospholipase D activity varied in different cell types. In addition, stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed in the leading edge than in the trailing edge of migrating HeLa cells. In MSC80 and OLN93 cells, which are stable cell lines derived from Schwann cells and oligodendrocytes, respectively, a higher ratio of diacylglycerol to PA levels was demonstrated in the membrane processes involved in myelination, compared to the cell body. We propose that the PA sensors reported here are valuable tools to unveil the role of PA in a variety of intracellular signaling pathways.

  2. Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view

    Directory of Open Access Journals (Sweden)

    Nathalie eLeborgne-Castel

    2014-12-01

    Full Text Available In order to ensure their physiological and cellular functions, plasma membrane (PM proteins must be properly conveyed from their site of synthesis, i.e. the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters.

  3. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vancini, Ricardo [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Kramer, Laura D. [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, NY (United States); Ribeiro, Mariana; Hernandez, Raquel [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States); Brown, Dennis, E-mail: dennis_brown@ncsu.edu [Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC (United States)

    2013-01-20

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  4. Flavivirus infection from mosquitoes in vitro reveals cell entry at the plasma membrane

    International Nuclear Information System (INIS)

    Vancini, Ricardo; Kramer, Laura D.; Ribeiro, Mariana; Hernandez, Raquel; Brown, Dennis

    2013-01-01

    Dengue and West Nile viruses are enveloped RNA viruses that belong to genus Flavivirus (family Flaviviridae) and are considered important mosquito-borne viral pathogenic agents worldwide. A potential target for intervention strategies is the virus cell entry mechanism. Previous studies of flavivirus entry have focused on the effects of biochemical and molecular inhibitors on viral entry leading to controversial conclusions suggesting that the process is dependent upon endocytosis and low pH mediated membrane fusion. In this study we analyzed the early events in the infection process by means of electron microscopy and immuno-gold labeling of viral particles during cell entry, and used as a new approach for infecting cells with viruses obtained directly from mosquitoes. The results show that Dengue and West Nile viruses may infect cells by a mechanism that involves direct penetration of the host cell plasma membrane as proposed for alphaviruses.

  5. 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

  6. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth.

    Directory of Open Access Journals (Sweden)

    Jiantao Zhang

    Full Text Available Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0 and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.

  7. Interactions between co-expressed Arabidopsis sucrose transporters in the split-ubiquitin system

    Directory of Open Access Journals (Sweden)

    Lalonde Sylvie

    2003-03-01

    Full Text Available Abstract Background The Arabidopsis genome contains nine sucrose transporter paralogs falling into three clades: SUT1-like, SUT2 and SUT4. The carriers differ in their kinetic properties. Many transport proteins are known to exist as oligomers. The yeast-based split ubiquitin system can be used to analyze the ability of membrane proteins to interact. Results Promoter-GUS fusions were used to analyze the cellular expression of the three transporter genes in transgenic Arabidopsis plants. All three fusion genes are co-expressed in companion cells. Protein-protein interactions between Arabidopsis sucrose transporters were tested using the split ubiquitin system. Three paralogous sucrose transporters are capable of interacting as either homo- or heteromers. The interactions are specific, since a potassium channel and a glucose transporter did not show interaction with sucrose transporters. Also the biosynthetic and metabolizing enzymes, sucrose phosphate phosphatase and sucrose synthase, which were found to be at least in part bound to the plasma membrane, did not specifically interact with sucrose transporters. Conclusions The split-ubiquitin system provides a powerful tool to detect potential interactions between plant membrane proteins by heterologous expression in yeast, and can be used to screen for interactions with membrane proteins as baits. Like other membrane proteins, the Arabidopsis sucrose transporters are able to form oligomers. The biochemical approaches are required to confirm the in planta interaction.

  8. 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

    proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized...... metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized...

  9. Redox enzymes in the plant plasma membrane and their possible roles

    DEFF Research Database (Denmark)

    Berczi, A.; Møller, I.M.

    2000-01-01

    Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K-1), all of which are likely to participate in redox...... protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b-type cytochrome. In co-operation with vitamin K-1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans-PM electron transport....

  10. Changes in plasma membrane state of thymocytes during spontaneous and radiation-induced leukemogenesis

    International Nuclear Information System (INIS)

    Gonta-Grabiec, K.

    1984-01-01

    Changes in plasma membrane properties characteristic for malignant cells were reviewed. Investigations of spontaneous (in AKR mice) and radiation-induced (in C57Bl) leukemogenesis were carried out; changes in properties of Na + , K + ATPase and alkaline phosphatase were characterized. On the basis of the results reported a pre-leukemic stage was distinguished, corresponding to the following features at the cellular level: increase in activity of alkaline phosphatase; decrease in relative activity of Na + , K + ATPase; decrease in efficiency of the Na + K + pump; decrease in cAMP content. 473 refs. (author)

  11. Advanced Fluorescence Microscopy Approaches to Understand the Dynamic Organization of the Plasma Membrane in Eukaryotes

    DEFF Research Database (Denmark)

    Ziomkiewicz, Iwona

    signaling in plants. Furthermore, it was established that ENODL9 clustering affects the organization of the PM and distribution of other PM proteins. Analysis of the phenotype of mutant lines revealed that ENODL9 has an important role for plant development and the adaptation to osmotic stress. This resulted......The plasma membrane (PM) is a physical barrier that defines the boundaries of a cell. It not only isolates the cell interior from the environment, but also enables cell communication and a selective exchange of solutes. To serve those contrasting functions, the PM has a dynamic structure consisting...

  12. Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

    DEFF Research Database (Denmark)

    Vind-Kezunovic, D.; Wojewodzka, U.; Gniadecki, R.

    2008-01-01

    ,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C-18:0), which specifically partitions to the liquid-disordered (L-d), non-raft phase, was also enriched in focal junctions and its mobility was slightly retarded. Cross-linking of GM(1) by CTB or raft aggregation by methyl......-p-cyclodextrin further decreased the recovery of DiI-C-18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L-d phase lipids. (c...

  13. Functional interactome of Aquaporin 1 sub-family reveals new physiological functions in Arabidopsis Thaliana

    Directory of Open Access Journals (Sweden)

    Mohamed Ragab Abdel Gawwad

    2013-09-01

    Full Text Available Aquaporins are channel proteins found in plasma membranes and intercellular membranes of different cellular compartments, facilitate the water flux, solutes and gases across the cellular plasma membranes. The present study highlights the sub-family plasma membrane intrinsic protein (PIP predicting the 3-D structure and analyzing the functional interactome of it homologs. PIP1 homologs integrate with many proteins with different plant physiological roles in Arabidopsis thaliana including; PIP1A and PIP1B: facilitate the transport of water, diffusion of amino acids and/or peptides from the vacuolar compartment to the cytoplasm, play a role in the control of cell turgor and cell expansion and involved in root water uptake respectively. In addition we found that PIP1B plays a defensive role against Pseudomonas syringae infection through the interaction with the plasma membrane Rps2 protein. Another substantial function of PIP1C via the interaction with PIP2E is the response to nematode infection. Generally, PIP1 sub-family interactome controlling many physiological processes in plant cell like; osmoregulation in plants under high osmotic stress such as under a high salt, response to nematode, facilitate the transport of water across cell membrane and regulation of floral initiation in Arabidopsis thaliana.

  14. Proteomic Identification of Annexins, Calcium-Dependent Membrane Binding Proteins That Mediate Osmotic Stress and Abscisic Acid Signal Transduction in Arabidopsis

    Science.gov (United States)

    Lee, Sumin; Lee, Eun Jung; Yang, Eun Ju; Lee, Ji Eun; Park, Ae Ran; Song, Won Hyun; Park, Ohkmae K.

    2004-01-01

    Comparative proteomic analysis of the Arabidopsis thaliana root microsomal fraction was performed to identify novel components of salt stress signaling. Among the salt-responsive microsomal proteins, two spots that increased upon salt treatment on a two-dimensional gel were identified as the same protein, designated annexin 1 (AnnAt1). Annexins comprise a multigene family of Ca2+-dependent membrane binding proteins and have been extensively studied in animal cells. AnnAt1 is strongly expressed in root but rarely in flower tissue. In this study, the results suggest that salt stress induces translocation from the cytosol to the membrane and potential turnover of existing protein. This process is blocked by EGTA treatment, implying that AnnAt1 functions in stress response are tightly associated with Ca2+. T-DNA insertion mutants of annAt1 and a different isoform, annAt4, displayed hypersensitivity to osmotic stress and abscisic acid (ABA) during germination and early seedling growth. The results collectively suggest that AnnAt1 and AnnAt4 play important roles in osmotic stress and ABA signaling in a Ca2+-dependent manner. PMID:15161963

  15. 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.

  16. 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.

  17. Plasma Biomarkers of Brain Injury as Diagnostic Tools and Outcome Predictors After Extracorporeal Membrane Oxygenation.

    Science.gov (United States)

    Bembea, Melania M; Rizkalla, Nicole; Freedy, James; Barasch, Noah; Vaidya, Dhananjay; Pronovost, Peter J; Everett, Allen D; Mueller, Gregory

    2015-10-01

    To determine if elevations in plasma brain injury biomarkers are associated with outcome at hospital discharge in children who require extracorporeal membrane oxygenation. Prospective observational study. Single tertiary-care academic center. Eighty children who underwent extracorporeal membrane oxygenation between June 2010 and December 2013. None. We measured six brain injury biomarkers (glial fibrillary acidic protein, monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2, neuron-specific enolase, S100b, intercellular adhesion molecule-5, and brain-derived neurotrophic factor) daily during extracorporeal membrane oxygenation, using an electrochemiluminescent multiplex assay. We recorded clinical, neuroimaging, and extracorporeal membrane oxygenation course data. We analyzed the association of biomarker concentrations with favorable versus unfavorable outcome at hospital discharge. Favorable outcome was defined as Pediatric Cerebral Performance Category 1, 2, or no change from baseline. Patients had a median age of 3 days (interquartile range, 1 d-10 mo), and 56% were male. Thirty-three of 80 (41%) had unfavorable outcome, and 22 of 70 (31%) had abnormal neuroimaging findings during or after extracorporeal membrane oxygenation. Peak concentrations were significantly higher in patients with unfavorable outcome than in those with favorable outcome for glial fibrillary acidic protein (p = 0.002), monocyte chemoattractant protein 1/chemokine (C-C motif) ligand 2 (p = 0.030), neuron-specific enolase (p = 0.006), and S100b (p = 0.015) and in patients with versus without abnormal neuroimaging findings for glial fibrillary acidic protein (p = 0.001) and intercellular adhesion molecule-5 (p = 0.001). The area under the receiver operator characteristic curve for unfavorable outcome was 0.73 for a noncollinear biomarker combination. After removing collinear biomarkers, the adjusted odds ratios for unfavorable outcome were 2.89 (95% CI, 1.09-7.73) for neuron

  18. 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.

  19. Plasma membrane cholesterol level and agonist-induced internalization of delta-opioid receptors; colocalization study with intracellular membrane markers of Rab family\

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Vošahlíková, Miroslava; Roubalová, Lenka; Parenti, M.; Mauri, M.; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-01-01

    Roč. 48, č. 4 (2016), s. 375-396 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 Keywords : cholesterol * plasma membrane * delta-opioid receptor * internalization * Rab proteins Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2016

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

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    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

  1. Analysis of the induction of the myelin basic protein binding to the plasma membrane phospholipid monolayer

    International Nuclear Information System (INIS)

    Zhang Lei; Hao Changchun; Feng Ying; Gao Feng; Lu Xiaolong; Li Junhua; Sun Runguang

    2016-01-01

    Myelin basic protein (MBP) is an essential structure involved in the generation of central nervous system (CNS) myelin. Myelin shape has been described as liquid crystal structure of biological membrane. The interactions of MBP with monolayers of different lipid compositions are responsible for the multi-lamellar structure and stability of myelin. In this paper, we have designed MBP-incorporated model lipid monolayers and studied the phase behavior of MBP adsorbed on the plasma membrane at the air/water interface by thermodynamic method and atomic force microscopy (AFM). By analyzing the pressure–area ( π – A ) and pressure–time ( π – T ) isotherms, univariate linear regression equation was obtained. In addition, the elastic modulus, surface pressure increase, maximal insertion pressure, and synergy factor of monolayers were detected. These parameters can be used to modulate the monolayers binding of protein, and the results show that MBP has the strongest affinity for 1,2-dipalmitoyl-sn-glycero-3- phosphoserine (DPPS) monolayer, followed by DPPC/DPPS mixed and 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) monolayers via electrostatic and hydrophobic interactions. AFM images of DPPS and DPPC/DPPS mixed monolayers in the presence of MBP (5 nM) show a phase separation texture at the surface pressure of 20 mN/m and the incorporation of MBP put into the DPPC monolayers has exerted a significant effect on the domain structure. MBP is not an integral membrane protein but, due to its positive charge, interacts with the lipid head groups and stabilizes the membranes. The interaction between MBP and phospholipid membrane to determine the nervous system of the disease has a good biophysical significance and medical value. (special topic)

  2. Membrane anchors effectively traffic recombinant human glucocerebrosidase to the protein storage vacuole of Arabidopsis seeds but do not adequately control N-glycan maturation.

    Science.gov (United States)

    He, Xu; Galpin, Jason D; Miao, Yansong; Jiang, Liwen; Grabowski, Gregory A; Kermode, Allison R

    2014-12-01

    Human glucocerebrosidase with vacuolar anchoring domains was targeted to protein storage vacuoles (PSVs) of Arabidopsis seeds, but unexpectedly via the Golgi complex. PSV-targeting to effectively avoid problematic N-glycans is protein dependent. Plant-specific N-glycosylation patterns elaborated within the Golgi complex are a major limitation of using plants to produce biopharmaceuticals as the presence of β1,2 xylose and/or α1,3 fucose residues on the recombinant glycoprotein can render the product immunogenic if administrated parenterally. A reporter protein fused to a vacuolar membrane targeting motif comprised of the BP-80 transmembrane domain (TMD), and the cytoplasmic tail (CT) of α-tonoplast intrinsic protein (α-TIP) is delivered to protein storage vacuoles (PSVs) of tobacco seeds by ER-derived transport vesicles that bypass the Golgi complex. This prompted us to investigate whether a pharmaceutical glycoprotein is targeted to PSVs using the same targeting sequences, thus avoiding the unwanted plant-Golgi-specific complex N-glycan modifications. The human lysosomal acid β-glucosidase (glucocerebrosidase; GCase) (EC 3.2.1.45) fused to the BP-80 TMD and α-TIP CT was produced in Arabidopsis thaliana wild-type (Col-0) seeds. The chimeric GCase became localized in PSVs but transited through the Golgi complex, as indicated by biochemical analyses of the recombinant protein's N-glycans. Our findings suggest that use of this PSV-targeting strategy to avoid problematic N-glycan maturation on recombinant therapeutic proteins is not consistently effective, as it is likely protein- and/or species-specific.

  3. Lpx1p links glucose-induced calcium signaling and plasma membrane H+-ATPase activation in Saccharomyces cerevisiae cells.

    Science.gov (United States)

    Castanheira, Diogo Dias; Santana, Eduardo Perovano; Godoy-Santos, Fernanda; Diniz, Raphael Hermano Santos; Faria-Oliveira, Fábio; Pereira, Renata Rebeca; Trópia, Maria José Magalhães; Castro, Ieso Miranda; Brandão, Rogelio Lopes

    2018-02-01

    In yeast, as in other eukaryotes, calcium plays an essential role in signaling transduction to regulate different processes. Many pieces of evidence suggest that glucose-induced activation of plasma membrane H+-ATPase, essential for yeast physiology, is related to calcium signaling. Until now, no protein that could be regulated by calcium in this context has been identified. Lpx1p, a serine-protease that is also involved in the glucose-induced activation of the plasma membrane H+-ATPase, could be a candidate to respond to intracellular calcium signaling involved in this process. In this work, by using different approaches, we obtained many pieces of evidence suggesting that the requirement of calcium signaling for activation of the plasma membrane H+-ATPase is due to its requirement for activation of Lpx1p. According to the current model, activation of Lpx1p would cause hydrolysis of an acetylated tubulin that maintains the plasma membrane H+-ATPase in an inactive state. Therefore, after its activation, Lpx1p would hydrolyze the acetylated tubulin making the plasma membrane H+-ATPase accessible for phosphorylation by at least one protein kinase. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    Science.gov (United States)

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  5. The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells.

    Science.gov (United States)

    Murphy, T. M.; Auh, C. K.

    1996-01-01

    Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells. PMID:12226208

  6. SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors.

    Science.gov (United States)

    Temkin, Paul; Lauffer, Ben; Jäger, Stefanie; Cimermancic, Peter; Krogan, Nevan J; von Zastrow, Mark

    2011-06-01

    Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.

  7. Parallel artificial liquid membrane extraction as an efficient tool for removal of phospholipids from human plasma.

    Science.gov (United States)

    Ask, Kristine Skoglund; Bardakci, Turgay; Parmer, Marthe Petrine; Halvorsen, Trine Grønhaug; Øiestad, Elisabeth Leere; Pedersen-Bjergaard, Stig; Gjelstad, Astrid

    2016-09-10

    Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Plasma membrane NADH oxidase of maize roots responds to gravity and imposed centrifugal forces

    Science.gov (United States)

    Bacon, E.; Morre, D. J.

    2001-01-01

    NADH oxidase activities measured with excised roots of dark-grown maize (Zea mays) seedlings and with isolated plasma membrane vesicles from roots of dark-grown maize oscillated with a regular period length of 24 min and were inhibited by the synthetic auxin 2,4-dichlorophenoxyacetic [correction of dichorophenoxyacetic] acid. The activities also responded to orientation with respect to gravity and to imposed centrifugal forces. Turning the roots upside down resulted in stimulation of the activity with a lag of about 10 min. Returning the sections to the normal upright position resulted in a return to initial rates. The activity was stimulated reversibly to a maximum of about 2-fold with isolated plasma membrane vesicles, when subjected to centrifugal forces of 25 to 250 x g for 1 to 4 min duration. These findings are the first report of a gravity-responsive enzymatic activity of plant roots inhibited by auxin and potentially related to the gravity-induced growth response. c2001 Editions scientifiques et medicales Elsevier SAS.

  9. The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux.

    Science.gov (United States)

    Sedgwick, Alanna; Olivia Balmert, M; D'Souza-Schorey, Crislyn

    2018-03-06

    Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease 1-4 . Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol. Copyright © 2018. Published by Elsevier Inc.

  10. The plasma membrane calcium pumps-The old and the new.

    Science.gov (United States)

    Zaidi, Asma; Adewale, Mercy; McLean, Lauren; Ramlow, Paul

    2018-01-10

    The plasma membrane Ca 2+ -ATPase (PMCA) pumps play a critical role in the maintenance of calcium (Ca 2+ ) homeostasis, crucial for optimal neuronal function and cell survival. Loss of Ca 2+ homeostasis is a key precursor in neuronal dysfunction associated with brain aging and in the pathogenesis of neurodegenerative disorders. In this article, we review evidence showing age-related changes in the PMCAs in synaptic plasma membranes (SPMs) and lipid raft microdomains isolated from rat brain. Both PMCA activity and protein levels decline progressively with increasing age. However, the loss of activity is disproportionate to the reduction of protein levels suggesting the presence of dysfunctional PMCA molecules in aged brain. PMCA activity is also diminished in post-mortem human brain samples from Alzheimer's disease and Parkinson's disease patients and in cell models of these neurodegenerative disorders. Experimental reduction of the PMCAs not only alter Ca 2+ homeostasis but also have diverse effects on neurons such as reduced neuritic network, impaired release of neurotransmitter and increased susceptibility to stressful stimuli, particularly to agents that elevate intracellular Ca 2+ [Ca 2+ ] i . Loss of PMCA is likely to contribute to neuronal dysfunction observed in the aging brain and in the development of age-dependent neurodegenerative disorders. Therapeutic (pharmacological and/or non-pharmacological) approaches that can enhance PMCA activity and stabilize [Ca 2+ ] i homeostasis may be capable of preventing, slowing, and/or reversing neuronal degeneration. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.

    Science.gov (United States)

    Khristov, Vladimir; Wan, Qin; Sharma, Ruchi; Lotfi, Mostafa; Maminishkis, Arvydas; Bharti, Kapil

    2018-01-01

    Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.

  12. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

    Science.gov (United States)

    Spalding, E. P.; Cosgrove, D. J.

    1989-01-01

    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  13. Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa

    Directory of Open Access Journals (Sweden)

    N. Prapaiwan

    2016-05-01

    Full Text Available Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05 than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05. In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

  14. PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

    Directory of Open Access Journals (Sweden)

    Oshima Kazuo

    2010-10-01

    Full Text Available Abstract Background The atypical cadherin protein cadherin 23 (CDH23 is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown. Results PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells. Conclusions PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.

  15. Alternative fates of newly formed PrPSc upon prion conversion on the plasma membrane.

    Science.gov (United States)

    Goold, Rob; McKinnon, Chris; Rabbanian, Samira; Collinge, John; Schiavo, Giampietro; Tabrizi, Sarah J

    2013-08-15

    Prion diseases are fatal neurodegenerative diseases characterised by the accumulation of misfolded prion protein (PrP(Sc)) in the brain. They are caused by the templated misfolding of normal cellular protein, PrP(C), by PrP(Sc). We have recently generated a unique cell system in which epitope-tagged PrP(C) competent to produce bona fide PrP(Sc) is expressed in neuroblastoma cells. Using this system we demonstrated that PrP(Sc) forms on the cell surface within minutes of prion exposure. Here, we describe the intracellular trafficking of newly formed PrP(Sc). After formation in GM1-enriched lipid microdomains at the plasma membrane, PrP(Sc) is rapidly internalised to early endosomes containing transferrin and cholera toxin B subunit. Following endocytosis, PrP(Sc) intracellular trafficking diverges: some is recycled to the plasma membrane via Rab11-labelled recycling endosomes; the remaining PrP(Sc) is subject to retromer-mediated retrograde transport to the Golgi. This pathway leads to lysosomal degradation, and we show that this is the dominant PrP(Sc) degradative mechanism in the early stages of prion infection.

  16. The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

    Science.gov (United States)

    Sgambato-Faure, Véronique; Xiong, Yuning; Berke, Joshua D.; Hyman, Steven E.; Strehler, Emanuel E.

    2013-01-01

    The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors, and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons. PMID:16554037

  17. 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.

  18. 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.

  19. Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

    Science.gov (United States)

    Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

    2017-08-01

    The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  20. Auxin-activated NADH oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

    Science.gov (United States)

    Morre, D. James; Morre, Dorothy M.; Ternes, Philipp

    2003-01-01

    The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14-17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.

  1. Proteomic analysis of plasma membrane and tonoplast from the leaves of mangrove plant Avicennia officinalis.

    Science.gov (United States)

    Krishnamurthy, Pannaga; Tan, Xing Fei; Lim, Teck Kwang; Lim, Tit-Meng; Kumar, Prakash P; Loh, Chiang-Shiong; Lin, Qingsong

    2014-11-01

    In order to understand the salt tolerance and secretion in mangrove plant species, gel electrophoresis coupled with LC-MS-based proteomics was used to identify key transport proteins in the plasma membrane (PM) and tonoplast fractions of Avicennia officinalis leaves. PM and tonoplast proteins were purified using two-aqueous-phase partitioning and density gradient centrifugation, respectively. Forty of the 254 PM proteins and 31 of the 165 tonoplast proteins identified were predicted to have transmembrane domains. About 95% of the identified proteins could be classified based on their functions. The major classes of proteins were predicted to be involved in transport, metabolic processes, defense/stress response, and signal transduction, while a few of the proteins were predicted to be involved in other functions such as membrane trafficking. The main classes of transporter proteins identified included H(+) -ATPases, ATP-binding cassette transporters, and aquaporins, all of which could play a role in salt secretion. These data will serve as the baseline membrane proteomic dataset for Avicennia species. Further, this information can contribute to future studies on understanding the mechanism of salt tolerance in halophytes in addition to salt secretion in mangroves. All MS data have been deposited in the ProteomeXchange with identifier PXD000837 (http://proteomecentral.proteomexchange.org/dataset/PXD000837). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

    Science.gov (United States)

    Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

    2016-05-01

    Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

  3. Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates.

    Science.gov (United States)

    Bienert, Gerd Patrick; Bienert, Manuela Désirée; Jahn, Thomas Paul; Boutry, Marc; Chaumont, François

    2011-04-01

    Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  4. Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

    DEFF Research Database (Denmark)

    Lagerholm, B. Christoffer; Andrade, Débora M.; Clausen, Mathias P.

    2017-01-01

    Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diff......Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below...... the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, ≥5 μm2 s-1, in the plasma membrane of live cells at very short length scales, ≈ 100 nm...

  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. 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.

  7. 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.

    2016-01-01

    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.

  8. 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.

  9. Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique.

    Science.gov (United States)

    Storey, Stephen M; Gibbons, Thomas F; Williams, Cecelia V; Parr, Rebecca D; Schroeder, Friedhelm; Ball, Judith M

    2007-06-01

    Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.

  10. 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

    2018-03-03

    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.

  11. Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens

    Science.gov (United States)

    Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.

    2012-01-01

    The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

  12. [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.

  13. The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum.

    Science.gov (United States)

    Li, Yan; Wang, Yannan; Zhang, Huan; Zhang, Qian; Zhai, Hong; Liu, Qingchang; He, Shaozhen

    2017-01-01

    SWEET (Sugars Will Eventually be Exported Transporter) proteins, a novel family of sugar transporters, mediate the diffusion of sugars across cell membranes and acts as key players in sucrose phloem loading. Manipulation of SWEET genes in plants leads to various effects on resistance to biotic and abiotic stresses due to disruption of sugar efflux and changes in sugar distribution. In this study, a member of the SWEET gene family, IbSWEET10 , was cloned from the sweet potato line ND98. mRNA expression analysis in sweet potato and promoter β-Glucuronidase analysis in Arabidopsis showed that IbSWEET10 is highly expressed in leaves, especially in vascular tissue. Transient expression in tobacco epidermal cells revealed plasma membrane localization of IbSWEET10, and heterologous expression assays in yeast indicated that IbSWEET10 encodes a sucrose transporter. The expression level of IbSWEET10 was significantly up-regulated in sweet potato infected with Fusarium oxysporum Schlecht. f. sp. batatas. Further characterization revealed IbSWEET10 -overexpressing sweet potato lines to be more resistant to F. oxysporum , exhibiting better growth after infection compared with the control; conversely, RNA interference (RNAi) lines showed the opposite results. Additionally, the sugar content of IbSWEET10 -overexpression sweet potato was significantly reduced, whereas that in RNAi plants was significantly increased compared with the control. Therefore, we suggest that the reduction in sugar content caused by IbSWEET10 overexpression is the major reason for the enhanced F. oxysporum resistance of the transgenic plants. This is the first report that the IbSWEET10 transporter contributes to the resistance of sweet potato to F. oxysporum . The IbSWEET10 gene has the great potential to be used for improving the resistance to F. oxysporum in sweet potato and other plants.

  14. Total internal reflection fluorescence (TIRF) microscopy for real-time imaging of nanoparticle-cell plasma membrane interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Moghimi, Seyed Moien

    2012-01-01

    fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching...... effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane....

  15. 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

    . 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....... Phosphopeptide enrichment by titanium dioxide chromatography followed by capillary liquid chromatography-tandem mass spectrometry allowed us to assign 703 unique phosphorylation sites in 376 phosphoproteins. Our experiments revealed that treatment of cell cultures with three different types of protein...

  16. 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.

  17. Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery

    International Nuclear Information System (INIS)

    McGuffee, L.J.; Little, S.A.; Mercure, J.V.; Skipper, B.J.; Wheeler-Clark, E.S.

    1990-01-01

    45Ca electron microscopic autoradiography was used to examine the effects of buffer composition and temperature on the distribution of calcium in rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the temperature of the bathing solution (25 degrees C or 34 degrees C). Krebs buffer at 34 degrees C gave the highest relative activity in the plasma membrane, sarcoplasmic reticulum, and mitochondria. Buffer and temperature had little effect on the relative activity of the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45 Ca in Krebs buffer at 34 degrees C. The results show that sarcoplasmic reticulum and plasma membrane are the primary sites of calcium accumulation during influx into these cells. Although the amount of 45 Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275 nm of the plasma membrane) is similar to the membrane alone and is lower than the sarcoplasmic reticulum alone

  18. A direct gateway into the extracellular space: Unconventional secretion of FGF2 through self-sustained plasma membrane pores.

    Science.gov (United States)

    Steringe