Patricia Gerbeau-Pissot

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (9)51.78 Total impact

  • Patricia Gerbeau-Pissot · Christophe Der · Markus Grebe · Thomas Stanislas
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    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.
    No preview · Article · Dec 2015 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: Lipid composition of plasma membrane (PM) and the corresponding Detergent-Insoluble Membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids so-called Glycosyl-Inositol-Phosphoryl-Ceramides (GIPCs). Using tobacco Bright Yellow-2 cell suspension and tobacco leaves (Nicotiana tabacum), evidences were provided that GIPCs represent up to 40 mol% of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated Very Long Chain Fatty Acid (VLCFA)-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35{plus minus}7nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols, and suggested a role for VLCFA in the interdigitation between the two PM composing-monolayers. The ins and outs of lipid asymmetry, raft formation and interdigitation in plant membrane biology are finally discussed.
    Full-text · Article · Oct 2015 · Plant physiology
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    ABSTRACT: The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids such as free- and conjugated- forms of phytosterols, and typical phytosphingolipids, such as glycosylinositolphosphoceramides (GIPC). This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with the environment-sensitive probe di-4-ANEPPDHQ. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation, and to organize their spatial distribution at the membrane surface. Conjugated-sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly GIPC, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free- and conjugated- phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Jan 2015 · Journal of Biological Chemistry
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    ABSTRACT: Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of BY2 tobacco suspension cells labeled with the fluorescent probe di-4-ANEPPDHQ. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of Fluorescence Recovery After Photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.
    Full-text · Article · Nov 2013 · Plant physiology
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    ABSTRACT: Lipids tend to organize in mono or bilayer phases in a hydrophilic environment. While they have long been thought to be incapable of coherent lateral segregation, it is now clear that spontaneous assembly of these compounds can confer microdomain organization beyond spontaneous fluidity. Membrane raft microdomains have the ability to influence spatiotemporal organization of protein complexes, thereby allowing regulation of cellular processes. In this review, we aim at summarizing briefly: (i) the history of raft discovery in animals and plants, (ii) the main findings about structural and signalling plant lipids involved in raft segregation, (iii) imaging of plant membrane domains, and their biochemical purification through detergent-insoluble membranes, as well as the existing debate on the topic. We also discuss the potential involvement of rafts in the regulation of plant physiological processes, and further discuss the prospects of future research into plant membrane rafts.
    Full-text · Article · Apr 2012 · Progress in lipid research
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    ABSTRACT: The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipid-enriched membrane rafts, emerges as a central regulatory mechanism governing physiological responses in various organisms. Over the past five years, plasma membrane located raft-like domains have been described in several plant species. The protein and lipid compositions of detergent-insoluble membranes, supposed to contain these domains, have been extensively characterised. Imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level at the plant plasma membrane, correlating detergent insolubility and membrane-domain localisation of presumptive raft proteins. Finally, the dynamic association of specific proteins with detergent-insoluble membranes upon environmental stress has been reported, confirming a possible role for plant rafts as signal transduction platforms, particularly during biotic interactions.
    No preview · Article · Sep 2011 · Current opinion in plant biology
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    ABSTRACT: The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.
    Preview · Article · Nov 2010 · Biochimica et Biophysica Acta
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    ABSTRACT: We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30 degrees C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-beta-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.
    Full-text · Article · Apr 2010 · Biochimica et Biophysica Acta
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    ABSTRACT: Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY-2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl-beta-cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl-conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid-phase heterogeneity in correlation with the disruption of phytosterol-rich domains. Methyl-beta-cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains.
    Preview · Article · Sep 2008 · The FASEB Journal

Publication Stats

158 Citations
51.78 Total Impact Points

Institutions

  • 2010-2013
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2011-2012
    • French National Institute for Agricultural Research
      Lutetia Parisorum, Île-de-France, France
  • 2008-2010
    • University of Burgundy
      Dijon, Bourgogne, France