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1. Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism

Author

Andreas P.M. Weber, Phillip Westhoff, Marianne Tardif, Denis Falconet, Erika Guglielmino, Dagmar Lyska, Benoit Gallet, Giovanni Finazzi, Gilles Curien, Clément Hallopeau, Michele Carone, Davide Dal Bo, Claire Remacle, Johan Decelle, Sabine Brugière, Myriam Ferro, Janina Janetzko, Light Photosynthesis & Metabolism (Photosynthesis), Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ)-Universität zu Köln = University of Cologne, Etude de la dynamique des protéomes (EDyP), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Photosymbiose, Institut de biologie structurale (IBS - UMR 5075), LIPID, Université de Liège, ARC grant (DARKMET proposal) for Concerted Research Actions (17/21-08), financed by the French Community of Belgium (Wallonia-Brussels Federation)., Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s Excellence Strategy – EXC-2048/1 – project ID 390686111, ANR-17-CE05-0029,MoMix,Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria(2017), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), European Project: 833184, ChloroMito, Martin-Laffon, Jacqueline, Modélisation de la Mixotrophie chez l'algue extrêmophile Galdieria sulphuraria - - MoMix2017 - ANR-17-CE05-0029 - AAPG2017 - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, CBH-EUR-GS - - CBH-EUR-GS2017 - ANR-17-EURE-0003 - EURE - VALID, Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID, Chloroplast and Mitochondria interactions for microalgal acclimation - ChloroMito - 833184 - INCOMING, Universität zu Köln-Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf]-Max Planck Institute for Plant Breeding Research (MPIPZ), and InBios/Phytosystems Research Unit, University of Liege

Subjects
Proteomics, 0106 biological sciences, 0301 basic medicine, photorespiration, Physiology, Heterotroph, Plant Science, Photosynthesis, 7. Clean energy, 01 natural sciences, Galdieria sulphuraria, Extremophiles, 03 medical and health sciences, mixotrophy, Total inorganic carbon, Botany, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, red algae, photosynthesis, biology, Chemistry, Research, RuBisCO, Heterotrophic Processes, Carbon Dioxide, Full Papers, 15. Life on land, Carbon, 030104 developmental biology, Rhodophyta, biology.protein, Photorespiration, Energy source, Mixotroph, 010606 plant biology & botany
Abstract

Summary Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon.Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2.Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose‐1,5‐bisphosphate carboxylase‐oxygenase (Rubisco) and decreasing photorespiration.We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

Published
2021

2. Unravelling hidden components of the chloroplast envelope proteome: opportunities and limits of better MS sensitivity

Author

Daniel Salvi, Norbert Rolland, Lucas Moyet, Imen Bouchnak, Marianne Tardif, Sabine Brugière, Marcel Kuntz, Sophie Le Gall, Dynamique du protéome et biogenèse du chloroplaste (ChloroGenesis), Physiologie cellulaire et végétale (LPCV), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), French National Research Agency (ANR) ANR-15-IDEX-02 ANR-10-LABX-49-01 ANR-10-INBS-08, Institut National de la Recherche Agronomique (INRA), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-15-IDEX-02,DATA@UGA,Grenoble Alpes Data Institute(2016), ANR-10- LABX-49-01,Labex GRAL,Labex GRAL, ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)

Subjects
Cell Extracts, Chloroplasts, Proteome, Arabidopsis thaliana, Arabidopsis, Plant Biology, Cellular organelles, Biochemistry, Chloroplast membrane, Subcellular Separation, Chloroplast, Cell fractionation, Analytical Chemistry, 03 medical and health sciences, Chloroplast Proteins, Organelle, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Plastid, Databases, Protein, Molecular Biology, 030304 developmental biology, 0303 health sciences, Subcellular analysis, Mass spectrometry, Chemistry, Arabidopsis Proteins, Subcellular localization, Research, 030302 biochemistry & molecular biology, Membrane Proteins, food and beverages, Proteins, Intracellular Membranes, Chloroplast envelope, Biogenesis, Subcellular Fractions
Abstract

By quantitatively comparing the proteomes of total leaf (crude cell extract) from Arabidopsis and purified chloroplast envelope fractions, this study makes available a novel parameter (calculated Enrichment Factor) for each putative envelope protein. This parameter provides important information to enable the more confident identification of genuine envelope components, distinguishing them from contaminants from other cellular/chloroplast compartments., Graphical Abstract Highlights Identification of previously undetected chloroplast envelope proteins. Up to date manual annotation of genuine (or shared) envelope components. New hypotheses for localizations, functions, interactions among cell compartments. A new resource of significant value to the broader plant science community., The chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling the exchange of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides etc.) of the plant cell. However, unraveling the contents of the chloroplast envelope proteome remains a difficult challenge; many proteins constituting this functional double membrane system remain to be identified. Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.4% of the whole cell proteome). In other words, most envelope proteins are so rare at the cell, chloroplast, or even envelope level, that they remained undetectable using targeted MS studies. Cross-contamination of chloroplast subcompartments by each other and by other cell compartments during cell fractionation, impedes accurate localization of many envelope proteins. The aim of the present study was to take advantage of technologically improved MS sensitivity to better define the proteome of the chloroplast envelope (differentiate genuine envelope proteins from contaminants). This MS-based analysis relied on an enrichment factor that was calculated for each protein identified in purified envelope fractions as compared with the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1269 proteins were detected in purified envelope fractions, of which, 462 could be assigned an envelope localization by combining MS-based spectral count analyses with manual annotation using data from the literature and prediction tools. Many of such proteins being previously unknown envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

Published
2019

3. The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

Author

Khawla Seddiki, Juliette Salvaing, Fabrice Rébeillé, Juliette Jouhet, Denis Falconet, Sabine Brugière, Yohann Couté, Eric Maréchal, Marcel Kuntz, Pierre-Henri Jouneau, Hubert Schaller, Antoine Jaussaud, Josselin Lupette, Marianne Tardif, Christian Morabito, Jean-Luc Putaux, LIPID, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de biologie moléculaire des plantes (IBMP), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Dynamique du protéome et biogenèse du chloroplaste (ChloroGenesis), Centre de Recherches sur les Macromolécules Végétales (CERMAV ), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA ), Modélisation et Exploration des Matériaux (MEM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Flagship program from the CEA High Commissioner, French National Research Agency (ANR) ANR-11-BTBR-0008 ANR-15-IDEX-02 ANR-10-INBS-08, ANR-11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-0002,UGA,IDEX UGA(2015), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherches sur les Macromolécules Végétales (CERMAV), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Laboratoire d'Etude des Matériaux par Microscopie Avancée (LEMMA), Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-11-BTBR-0008/11-BTBR-0008,OCEANOMICS,Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques(2011), ANR-15-IDEX-02,DATA@UGA,Grenoble Alpes Data Institute(2016), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Jouneau, Pierre-Henri, Biotech - Bioressources - Biotechnologies et bioressources pour la valorisation des écosystèmes marins planctoniques - - OCEANOMICS2011 - ANR-11-BTBR-0008 - BTBR - VALID, IDEX UGA - - UGA2015 - ANR-15-IDEX-0002 - IDEX - VALID, and Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID

Subjects
0106 biological sciences, 0301 basic medicine, Betaine lipid, Phaeodactylum, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], 01 natural sciences, Ribosome, Triacylglycerol, [SDV.BV.BOT] Life Sciences [q-bio]/Vegetal Biology/Botanics, Histones, 03 medical and health sciences, Lipid droplet, Protein biosynthesis, ERAD pathway, Phaeodactylum tricornutum, Plastid, Secondary plastid, ComputingMilieux_MISCELLANEOUS, Diatoms, biology, Chemistry, Endoplasmic reticulum, fungi, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Lipid droplets, Carotenoids, 030104 developmental biology, Biochemistry, Proteome, Agronomy and Crop Science, 010606 plant biology & botany
Abstract

International audience; Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previously validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl-beta-alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of beta-carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function includes protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.

Published
2019

4. Optical tweezing using tunable optical lattices along a few-mode silicon waveguide

Author

F. de Fornel, Emmanuel Picard, Jean-Baptiste Jager, Benoit Cluzel, Marianne Tardif, Christophe Pin, Emmanuel Hadji, Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Bourgogne (UB)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), Silicon Nanoelectronics Photonics and Structures (SiNaps), PHotonique, ELectronique et Ingénierie QuantiqueS (PHELIQS), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service de Physique des Matériaux et Microstructures (SP2M - UMR 9002), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ANR-11-LABX-0001,ACTION,Systèmes intelligents intégrés au cœur de la matière(2011), Laboratoire Interdisciplinaire Carnot de Bourgogne [Dijon] (LICB), and Université de Bourgogne (UB)-Université de Technologie de Belfort-Montbeliard (UTBM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Silicon, Materials science, Optical Tweezers, Biomedical Engineering, Nanophotonics, Holography, chemistry.chemical_element, Physics::Optics, Bioengineering, 02 engineering and technology, Trapping, Models, Biological, 01 natural sciences, Biochemistry, Waveguide (optics), law.invention, 010309 optics, law, Lab-On-A-Chip Devices, 0103 physical sciences, Tweezers, Light beam, Particle Size, business.industry, General Chemistry, 021001 nanoscience & nanotechnology, Microspheres, [SPI.ELEC]Engineering Sciences [physics]/Electromagnetism, chemistry, Optical tweezers, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, Nanoparticles, Optoelectronics, 0210 nano-technology, business
Abstract

International audience; Fourteen years ago, optical lattices and holographic tweezers were considered as a revolution, allowing for trapping andmanipulating multiple particles at the same time using laser light. Since then, near-field optical forces have arousedtremendous interest as they enable efficient trapping of a wide range of objects, from living cells to atoms, in integrateddevices. Yet, handling at will multiple objects using a guided light beam remains a challenging task for current on-chipoptical trapping techniques. We demonstrate here on-chip optical trapping of dielectric microbeads and bacteria usingone-dimensional optical lattices created by near-field mode beating along a few-mode silicon nanophotonic waveguide.This approach allows not only for trapping a large number of particles in periodic trap arrays with various geometries, butalso for manipulating them via diverse transport and repositioning techniques. Near-field mode-beating optical latticesmay be readily implemented in lab-on-a-chip devices, addressing numerous scientific fields ranging from bio-analysis tonanoparticle processing.

Published
2018

5. AtMic60 Is Involved in Plant Mitochondria Lipid Trafficking and Is Part of a Large Complex

Author

Denis Falconet, Michael R. Wozny, Juliette Jouhet, William A. Prinz, Maryse A. Block, Sabine Brugière, Morgane Michaud, Eric Maréchal, Marianne Tardif, Valérie Gros, Myriam Ferro, Jaideep Mathur, Alexandre Toulmay, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Department of Molecular and Cellular Biology, University of Guelph, EMBO (ASTF-638-2014), ANR-12-JSV2-0001,ChloroMitoLipid,Export de galactolipides des chloroplastes vers les mitochondries(2012), ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR-10-LABX-0004,CeMEB,Mediterranean Center for Environment and Biodiversity(2010), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), ANR-10-INBS-08-01/10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), ANR–10–LABEX–04 ,GRAL,Labex, Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Arabidopsis, TIM/TOM complex, Mitochondrial Membrane Transport Proteins, Galactoglycerolipids, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Mitochondrial membrane transport protein, chemistry.chemical_compound, Organelle, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Digalactosyldiacylglycerol, Phosphate starvation, Membrane biogenesis, Phospholipids, Phosphatidylethanolamine, biology, Agricultural and Biological Sciences(all), Biochemistry, Genetics and Molecular Biology(all), Arabidopsis Proteins, Endoplasmic reticulum, Lipids transfer, Plant, Lipid Metabolism, Membrane contact site, Cell biology, Mitochondria, Protein Transport, 030104 developmental biology, Membrane, Biochemistry, chemistry, Lipoprotein complex, biology.protein, General Agricultural and Biological Sciences
Abstract

International audience; The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.

Published
2016

6. Inhibitory effects of a dominant-interfering form of the Rho-GTPase Cdc42 in the chemoattractant-elicited signaling pathways leading to NADPH oxidase activation in differentiated HL-60 cells

Author

Laurence Braun, François Boulay, Marianne Tardif, and Marie-Josèphe Rabiet

Subjects
Transcriptional Activation, Immunology, Guanosine, HL-60 Cells, GTPase, Biochemistry, chemistry.chemical_compound, Transactivation, Humans, Promoter Regions, Genetic, cdc42 GTP-Binding Protein, NADPH oxidase, biology, Superoxide, NADPH Oxidases, Cell Biology, Hematology, Tetracycline, Cell biology, Enzyme Activation, chemistry, biology.protein, Guanine nucleotide exchange factor, Signal transduction, Nicotinamide adenine dinucleotide phosphate, Signal Transduction
Abstract

A tetracycline-controlled expression system was adapted to the human promyelocytic HL-60 cell line by placement of the transactivator (tTA-off) sequence under the control of the human EF-1α promoter region. Constitutively active and dominant-inhibitory forms of Cdc42 (Cdc42V12 and Cdc42N17, respectively) were conditionally expressed in this system. The expression of Cdc42V12 had no marked effect on chemoattractant-mediated superoxide production, corroborating previous results indicating that the guanosine 5′-triphosphate (GTP)–bound form of Cdc42 is ineffective in directly activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in a cell-free system. However, the N17 mutant potently inhibited chemoattractant-induced superoxide production. The expression of Cdc42N17 interfered with the GTP-loading of Rac and Ras and with the activation of the MAP-kinase pathway. A drastic reduction of chemoattractant-induced inositol-1,4,5-trisphosphate formation and calcium mobilization was observed, corroborating previous in vitro study results identifying PLCβ2 as a Rac/Cdc42 effector. Cdc42N17 was also found to inhibit the translocation of Ras-GRF2, a guanine nucleotide exchange factor for Ras and Rac but not for Cdc42. Thus, the dominant-inhibitory mutant Cdc42N17 was found to interfere at multiple levels in the signaling pathways. The pleiotropic inhibitory effects of Cdc42N17 illustrate the potential pitfalls of using dominant-inhibitory proteins to study the function of Ras-family GTPases. In this regard, a number of conclusions drawn from the use of dominant-inhibitory mutants in myeloid cells might have to be reconsidered.

Published
2002

7. Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

Author

François Boulay, Richard D. Ye, Joseph R. Woska, Faripour Forouhar, Barbara Jean Bormann, Darlene Sodja, Marianne Tardif, Philip M. Murphy, Margaret L. Grunnet, and Elmer L. Becker

Subjects
Male, Cell type, medicine.medical_specialty, Histology, Receptors, Peptide, Enteroendocrine cell, Biology, Pathology and Forensic Medicine, Neuroblastoma, chemistry.chemical_compound, Immune system, Antigen, Internal medicine, Tumor Cells, Cultured, medicine, Humans, Antigens, Receptors, Immunologic, Receptor, Antiserum, Binding Sites, Cell Biology, N-Formylmethionine leucyl-phenylalanine, Immunohistochemistry, Receptors, Formyl Peptide, Cell biology, N-Formylmethionine Leucyl-Phenylalanine, Endocrinology, chemistry, Organ Specificity, biology.protein, Female, Antibody, Protein Binding
Abstract

The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.

Published
1998

8. Characterization of chloroplastic fructose 1,6-bisphosphate aldolases as lysine-methylated proteins in plants

Author

Stéphane Ravanel, Morgane Mininno, Sheng Ma, Myriam Ferro, Marianne Tardif, Claude Alban, Virginie Pautre, Sabine Brugière, Annabelle Gilgen, Laboratoire de physiologie cellulaire végétale (LPCV), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Etude de la dynamique des protéomes (EDyP), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Etude de la dynamique des protéomes (EDyP ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Department of Plant Biology INRA, Cluster 9 of the Region Rhone-Alpes, French National Research Agency [SVSE6], Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Martin-Laffon, Jacqueline, Laboratoire d'étude de la dynamique des protéomes (LEDyP), and Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0106 biological sciences, Chloroplasts, Fructose 1,6-bisphosphate, ribulose bisphosphate carboxylase, Arabidopsis thaliana, substrate specificity, Arabidopsis, Plant Biology, Fructose-bisphosphate aldolase, plant, 01 natural sciences, Biochemistry, protein lysine methyltransferase, chemistry.chemical_compound, kinetic properties, Fructose-Bisphosphate Aldolase, Protein methylation, 0303 health sciences, biology, food and beverages, chloroplast, enzyme kinetics, plant biochemistry, protein methylation, s adenosylmethionine, aldolase, rubisco, Chloroplast, protéine, plante, inorganic chemicals, LSMT, Ribulose-Bisphosphate Carboxylase, 6-bisphophate aldolase, complex mixtures, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Pisum sativum, 030304 developmental biology, Ribulose, RuBisCO, Aldolase A, fungi, Peas, Cell Biology, légume, biology.organism_classification, fructose 1, enzyme, chemistry, biology.protein, bacteria, methylation, Protein Processing, Post-Translational, metabolism, 010606 plant biology & botany
Abstract

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; In pea (Pisum sativum), the protein-lysine methyltransferase (PsLSMT) catalyzes the trimethylation of Lys-14 in the large subunit (LS) of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme catalyzing the CO(2) fixation step during photosynthesis. Homologs of PsLSMT, herein referred to as LSMT-like enzymes, are found in all plant genomes, but methylation of LS Rubisco is not universal in the plant kingdom, suggesting a species-specific protein substrate specificity of the methyltransferase. In this study, we report the biochemical characterization of the LSMT-like enzyme from Arabidopsis thaliana (AtLSMT-L), with a focus on its substrate specificity. We show that, in Arabidopsis, LS Rubisco is not naturally methylated and that the physiological substrates of AtLSMT-L are chloroplastic fructose 1,6-bisphosphate aldolase isoforms. These enzymes, which are involved in the assimilation of CO(2) through the Calvin cycle and in chloroplastic glycolysis, are trimethylated at a conserved lysyl residue located close to the C terminus. Both AtLSMT-L and PsLSMT are able to methylate aldolases with similar kinetic parameters and product specificity. Thus, the divergent substrate specificity of LSMT-like enzymes from pea and Arabidopsis concerns only Rubisco. AtLSMT-L is able to interact with unmethylated Rubisco, but the complex is catalytically unproductive. Trimethylation does not modify the kinetic properties and tetrameric organization of aldolases in vitro. The identification of aldolases as methyl proteins in Arabidopsis and other species like pea suggests a role of protein lysine methylation in carbon metabolism in chloroplasts.

Published
2012

9. Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA

Author

Pierre V. Vignais, Marianne Tardif, François Boulay, and Laurence Brouchon

Subjects
G protein, Molecular Sequence Data, Biophysics, Peptide, Biology, Transfection, Biochemistry, Cell Line, Complementary DNA, Animals, Humans, Amino Acid Sequence, Cloning, Molecular, Receptors, Immunologic, Receptor, Molecular Biology, Gene Library, chemistry.chemical_classification, Expression vector, COS cells, Formyl peptide receptor, Base Sequence, Superoxide Dismutase, cDNA library, DNA, Cell Biology, Receptors, Formyl Peptide, Molecular biology, N-Formylmethionine Leucyl-Phenylalanine, chemistry
Abstract

N-formyl-methionyl peptides are powerful chemoattractants which bind to specific receptors on the neutrophil plasma membrane. A cDNA library from HL-60 cells, differentiated into granulocytes highly responsive to N-formyl-methionyl peptides, was constructed in the COS cell expression vector CDM8. A cDNA clone was isolated that conferred to COS cells the ability to bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys. The transfected COS cells displayed two classes of binding sites with Kd values of 1–5nM and 5–10 nM, respectively. The cDNA was 1.9 kb long with a 1050 bp open reading frame encoding a 350 residue protein. The hydropathy plot analysis revealed seven hydrophobic segments, a pattern quite similar to that of G protein-coupled receptors.

Published
1990

10. Phagocyte chemoattractant receptors

Author

François Boulay, L. Brouchon, E. Giannini, N. Naik, and Marianne Tardif

Subjects
MAPK/ERK pathway, Chemokine, Phagocyte, Molecular Sequence Data, Complement C5a, General Biochemistry, Genetics and Molecular Biology, Chemokine receptor, History and Philosophy of Science, medicine, Humans, Amino Acid Sequence, Platelet Activating Factor, Receptor, Phagocytes, biology, Chemotactic Factors, Chemistry, General Neuroscience, Chemotaxis, Cell biology, medicine.anatomical_structure, biology.protein, Phosphorylation, Receptors, Chemokine, Signal transduction, Signal Transduction
Abstract

Myeloid cells are attracted and activated by a variety of chemoattractants that bind to G protein-coupled receptors. In the past few years, the receptors for the classical chemoattractants (fMLF, C5a, PAF) and the chemotactic cytokines, known as C-X-C and C-C chemokines, have been cloned from myeloid cells. This review briefly describes recent advances in structure-function relationships of chemotactic receptors in human leukocytes as well as activation of signaling pathways and regulation of receptor function. In neutrophils, the binding of chemoattractants mainly activates the Gi2 protein inducing PIP2 hydrolysis and activation of the MAP kinase pathway. The C-C chemokine receptor, CC CKR5, and a chemokine receptor homologue, named fusin, have been shown to be the major cofactors for HIV-1 entry in macrophages and T cells. Recent studies suggest that the phosphorylation of chemoattractant receptors is a key event that regulates their biological function.

Published
1998

11. Actin polymerization induced by GTP gamma S in permeabilized neutrophils is induced and maintained by free barbed ends

Author

Marianne Tardif, Sherry Huang, Daniel Safer, Tim Redmond, Sally H. Zigmond, and M. Pring

Subjects
Cell Membrane Permeability, Time Factors, Cytochalasin B, Neutrophils, Phalloidine, Guanosine, macromolecular substances, In Vitro Techniques, Biochemistry, chemistry.chemical_compound, Bacterial Proteins, Animals, Deoxyribonuclease I, Cytochalasin, Molecular Biology, Actin, Fluorescent Dyes, biology, Depolymerization, Rhodamines, Cell Biology, Actins, Thymosin, Kinetics, chemistry, Profilin, Polymerization, Guanosine 5'-O-(3-Thiotriphosphate), Streptolysins, biology.protein, Biophysics, Streptolysin, Rabbits, Mathematics
Abstract

To address the mechanisms through which agonists stimulate actin polymerization, we examined the roles of monomer sequestering proteins and free barbed ends on actin polymerization induced by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) in neutrophils permeabilized with streptolysin O. Addition of profilin (without GTP gamma S) caused a net decrease in F-actin. Thus, merely making profilin available in the cell was not sufficient to induce actin polymerization. On the other hand, addition of profilin hardly affected the polymerization induced by GTP gamma S, while thymosin beta 4 or DNase I decreased this polymerization. These data suggested that GTP gamma S induced polymerization by increasing the availability of barbed ends. In the presence of cytochalasin B, profilin did inhibit polymerization induced by GTP gamma S, demonstrating that GTP gamma S did not inhibit profilin's monomer sequestering ability. The F-actin induced by GTP gamma S was not limited by a time-dependent loss of G-actin or G-proteins from permeabilized cells since, following stimulation with suboptimal concentrations of GTP gamma S, addition of more GTP gamma S induced further polymerization. Barbed ends remained free after F-actin reached plateau since (a) cytochalasin B caused depolymerization of induced F-actin and (b) profilin did not depolymerize induced F-actin unless the cells were first treated with cytochalasin to cap barbed ends. The data indicate that GTP gamma S maintains an increased level of F-actin by keeping at least a few barbed ends available for polymerization.

Published
1995

12. Activation of superoxide radical anion generating oxidase of bovine neutrophils in a cell-free system. Interaction of a cytosolic factor with the plasma membrane and control by G nucleotides

Author

Erzsébet Ligeti, Pierre V. Vignais, and Marianne Tardif

Subjects
chemistry.chemical_classification, Oxidase test, biology, Chemistry, Serum albumin, Plasma protein binding, Biochemistry, Cell-free system, chemistry.chemical_compound, Cytosol, Cytoplasm, biology.protein, Arachidonic acid, Nucleotide
Abstract

Activation of the O2.- -generating oxidase of bovine neutrophils was studied in a cell-free system, consisting of a particulate fraction enriched in plasma membrane, cytosol, arachidonic acid, and the non-hydrolyzable nucleotide GTP-gamma-S. Activation of the membrane-bound oxidase was accompanied by the disappearance of the activating factor from the cytosol. Above a cytosol to membrane ratio of 25, the excess of added cytosolic factor remained in active state in the soluble fraction. The process could be partially reversed by serum albumin. Disappearance of the cytosolic factor was promoted by unsaturated long-chain fatty acids, but not by saturated ones, and occurred not only in the presence of GTP-gamma-S but also in the presence of GDP-beta-S or in the absence of Mg ions, although in the latter cases activation of O2.- production was seriously impaired. This suggests that the disappearance of the activating factor from the cytosol and the triggering effect of GTP-gamma-S are related, but distinct, events in the oxidase activation process. The disappearance of the activating factor from cytosol can be explained by translocation of the cytosolic factor to the membrane fraction. Yet under some conditions, including the presence of GDP-beta-S or EDTA, inactivation was prevailing and could be an alternative explanation for the results. Specific binding of radiolabeled GTP-gamma-S could be demonstrated both in the membrane and in the cytosolic fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Published
1989

13. INDUCTION OF ACTIN POLYMERIZATION IN PERMEABILIZED NEUTROPHILS - ROLE OF ATP

Author

Marianne Tardif, Sally H. Zigmond, and Tim Redmond

Subjects
Cell Membrane Permeability, GTP', Phalloidin, Neutrophils, Polymers, Guanosine, macromolecular substances, Pertussis toxin, Biochemistry, chemistry.chemical_compound, Adenosine Triphosphate, Bacterial Proteins, medicine, Animals, Ascitic Fluid, Molecular Biology, Actin, Guanylyl Imidodiphosphate, Chemotactic Factors, Dose-Response Relationship, Drug, Apyrase, Nucleotides, Cell Biology, Adenosine, Actins, chemistry, Guanosine 5'-O-(3-Thiotriphosphate), Streptolysins, Biophysics, Phosphorylation, Guanosine Triphosphate, Rabbits, Oligopeptides, medicine.drug, Signal Transduction
Abstract

We have used streptolysin-O (SO)-permeabilized neutrophils to investigate the signal transduction pathway through which chemoattractants induce actin polymerization. Chemoattractants stimulate phosphorylation of various proteins and lipids but whether these phosphorylations are required for actin polymerization is not known. Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to SO-permeabilized neutrophils induced a doubling of the F-actin. This induction of F-actin, assayed by TRITC-labeled phalloidin binding, did not require the addition of ATP. Neither addition of apyrase to deplete residual ATP nor addition of ADP or UDP to compete with residual endogenous ATP inhibited significantly the GTP gamma S-induced polymerization. Addition of ATP on its own caused no increase in F-actin and did not affect the time course or concentration dependence of GTP gamma S-induced F-actin. Addition of ATP did increase the maximal amount of F-actin induced by GTP gamma S by about 20%. N-Formylnorleucylleucylphenalanine (formyl-peptide) in the presence of GTP, but not in its absence, also stimulated an increase in F-actin in SO-permeabilized cells. The F-actin induced by formyl-peptide plus GTP was inhibited by pertussis toxin. The induction did not require addition of ATP and addition of ADP to compete with residual ATP only slightly decreased the level of actin. However, addition of UDP significantly reduced the response to formyl-peptide plus GTP. Addition of ATP enhanced the increase in F-actin induced by optimal concentrations of GTP with formyl-peptide. ATP also lowered the apparent Km for GTP, but not for N-formyl peptide. The non-hydrolyzable ATP analog, adenosine 5'-(beta, gamma-imino)triphosphate, did not enhance the actin polymerization. Rather its presence inhibited the response induced by formyl-peptide plus GTP. The data suggest that actin polymerization can be induced by GTP gamma S in an manner that is largely ATP-independent. A role for ATP cannot be ruled out in the induction of actin polymerization by formyl-peptide plus GTP.

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