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24, chemin de Borde Rouge –Auzeville – CS52627
31326 Castanet Tolosan CEDEX - France

Dernière mise à jour : Mai 2018

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Practical Training

July 4-6, 11-13

The second half of both weeks of the school is devoted to practical training workshops (3 to 6 persons per workshop).

Each attendee will follow 2 workshops amongst the following 8 possibilities :

 W1: Characterisation of ion uptake in plants using stable isotopes: a case study with NO3-
          Alain Gojon, Pascal Tillard (BPMP, Montpellier)

This workshop will illustrate how the use of the non-radioactive 15N tracer, and its analysis by Isotope Ratio Mass Spectrometry to quantify ion fluxes in the plant allow to delineate plant adaptive response to nitrogen availability. The experiments will aim at characterizing Arabidopsis seedlings (WT and mutants) grown in vitro on a wide range of NO3- concentrations. Root NO3- uptake capacity will be determined using 15N-labelled NO3- and quantification, by Mass Spectrometry.

W2: ROS and protein subcellular dynamics imaging in plants
          Doan Luu, Lionel Verdoucq (BPMP, Montpellier)

In this workshop we will use wild type and transgenic Arabidopsis lines to study the cellular signalling and subcellular dynamics. Firstly, by using cell imaging techniques, we will analyze Reactive Oxygen Species (H2O2) cytosolic accumulation in Arabidopsis guard cells in response to a biotic signal. Here, HyPer sensor will be used. Secondly, we will analyze the dynamics of subcellular localization of membrane markers upon abiotic stress, by means of confocal imaging with photoconversion and fluorescence recovery after photobleaching. Here, plasma membrane aquaporins stably expressed in root cells will be used as prototypes of membrane proteins. Time will also be dedicated to explain the different microscope settings, the advantages and limitation of the methods.

W3: Super resolution analysis of protein diffusion and clustering in the membrane
          Carine Alcon, Alexandre Martinière (BPMP, Montpellier), Jean-Bernard Fiche (CBS, Montpellier)

Within membrane, proteins are organized in nanodomains. This particular space regulation is required for many aspects of biology, especially cell signalling. We will use super resolution microscopy (sptPALM) to quantify protein clustering and diffusion at molecular scale in response to various elicitors. Part of this workshop will be also devoted to data analysis (track reconstruction, quantification of diffusion coefficient and local density).

 W4: Voltage-clamp analyses of transporters and voltage-gated channels ("whole-cell" current recordings), as examples of non-rectifying and rectifying conductances
          Martin Boeglin, Claire Corratgé-Faillie, Anne Aliénor Véry (BPMP, Montpellier)

Addressed topics will be oocyte microinjection for heterologous expression of an ion transport system, ion current recordings using the voltage-clamp technique and data analysis to extract functional properties.

 W5: Patch-clamp analysis of a voltage-gated channel: single channel recording
          Martin Boeglin, Claire Corratgé-Faillie, Anne Aliénor Véry (BPMP, Montpellier)

Addressed topics will be oocyte microinjection, ion current recordings using the patch-clamp technique and data analysis.

 W6: Initiation to proteomics and phosphoproteomics
          Nathalie Berger, Vincent Demolombe, Sonia Hem, Valérie Rofidal (Plateforme de spectrométrie de masse pour la protéomique, BPMP, Montpellier)

This workshop will address the use of mass spectrometry to identify and quantify proteins in their native and phosphorylated forms. During the first day, an introduction to principles in mass spectrometry for protein identification will be provided. Then a proteomic experiment will be conducted including protein digestion, phosphopeptides enrichment and peptides injection in the mass spectrometer. During the second day, raw data will be processed to identify and quantify through the use of a pipeline of dedicated softwares. A critical analysis of (phospho)- peptide spectrum will also be provided.

 W7: Hydraulic conductivity of membranes, cells and organs
          Yann Boursiac, Christophe Maurel, Lionel Verdoucq (BPMP, Montpellier)

The aim of this practical is to exemplify the specificities and limitations of water transport assays in various membrane, cell and organ systems. Water transport in purified membrane vesicles or wall-deprived cell systems (Xenopus oocytes, isolated plant protoplasts) will be monitored using various types of osmotic swelling or shrinking assays. A cell pressure probe will be manipulated to address the principles of turgor and hydrostatic relaxation measurements in plant cells. Finally, pressure chamber measurements in excised roots and rosettes will be performed. All assays will be designed to investigate typical features of aquaporin regulation.

 W8: Yeast as a tool: characterisation of ion transport systems by complementation of mutant strains and 2-hybrid tests for revealing protein-protein interactions
        Léon Dirick, Florence Vignols (BPMP, Montpellier)

The aim of this workshop is to illustrate the methodological aspect of yeast as a tool to study (i) functional complementation of yeast mutants  by homologous plant genes, as well as (ii) to test physical interactions occurring in vivo between potential protein interactors.