title: Ratiometric Fluorescence Live Imaging Analysis of Membrane Lipid Order in Arabidopsis Mitotic Cells Using a Lipid Order-Sensitive Probe.
abstract: Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana.
authors.affiliation: Unité Mixte de Recherche 1347 Agroécologie, Equipe de Recherche Labélisée 6300 Centre National de la Recherche Scientifique, Université de Bourgogne, BP 86510, 21000, Dijon, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Gerbeau-Pissot
authors.affiliation: Unité Mixte de Recherche 1347 Agroécologie, Equipe de Recherche Labélisée 6300 Centre National de la Recherche Scientifique, Université de Bourgogne, BP 86510, 21000, Dijon, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Der
authors.affiliation: Pflanzenphysiologie, Institut für Biochemie und Biologie, Universität Potsdam, Karl-Liebknecht-Str. 24-25, Haus 20, Potsdam-Golm, 14476, Germany.|Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187, Umea, Sweden.
authors.forename: Markus
authors.initials: M
authors.lastname: Grebe
authors.affiliation: Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, 90187, Umea, Sweden. Thomas.stanislas@ens-lyon.fr.|Laboratoire de Reproduction de développement des plantes, INRA, CNRS, ENS Lyon, UCB Lyon 1, Université de Lyon, 46 Allée d'Italie, 69364, Lyon, Cedex 07, France. Thomas.stanislas@ens-lyon.fr.|Laboratoire Joliot-Curie, Laboratoire de Physique, CNRS, ENS Lyon, UCB Lyon 1, Université de Lyon, 46 Allée d'Italie, 69364, Lyon, Cedex 07, France. Thomas.stanislas@ens-lyon.fr.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stanislas
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000648:ultrastructure; D018929:Cell Culture Techniques; D005456:Fluorescent Dyes / Q000032:analysis*; D008563:Membrane Lipids / Q000032:analysis*; D021962:Membrane Microdomains / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D008938:Mitosis; D061848:Optical Imaging / Q000379:methods; D018517:Plant Roots / Q000166:cytology / Q000648:ultrastructure; D011726:Pyridinium Compounds / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D008563:Membrane Lipids; D011726:Pyridinium Compounds; C541550:di-4-ANEPPDHQ
keywords: Arabidopsis roots; Arabidopsis suspension cells; Cell plate; Di-4-ANEPPDHQ; Membrane order; Mitosis protocols