Programme (pre)
Preliminary Course Programme | Week One
Second Week: “The Advanced Techniques”
In the second week of the course, the model organisms used will be extended to Salmonella enterica serovar Typhimurium and Entamoeba histolytica. The students will be divided in 4 groups that will be able to select specialized practicals according to their particular technical interests and background. The many practicals will be focused on microscopy techniques, but cell sorting and compartment fractionation will be also addressed.
Practical A “Dynamic Analysis of the Clathrin-Independent Endocytosis of the Interleukin 2 Receptor using Total Internal Reflection Fluorescence (TIRF) Microscopy”: TIRF allows the selective study of phenomena taking place in a thin region near the plasma membrane –usually less than 100 nm in depth. This technique allows the analysis of the internalization of the interleukin 2-receptor, which occurs by a clathrin-independent endocytosis phenomenon (Sauvonnet et al. 2005. J. Cell Biol. 168: 155). |
Practical B “Activation of the PI3 Kinase-Dependent Pathway at the Plasma Membrane of Target Cells Upon Stimulation with InlB Using Fluorescence Resonance Energy Transfer (FRET) Microscopy”: FRET is a powerful technique that allows the investigation of molecular interactions with an optical microscope well beyond the theoretical resolution limit of light, when the proximity of a suitable pair of spectrally distinct fluorescent molecules is sufficiently close (between 1 and 10 nm) for a radiationless transfer of energy from an excited fluorophore (donor) to the second fluorophore (acceptor). Using this technique we will monitor the activation of the PI3 kinase pathway upon stimulation by InlB of Listeria monocytogenes using the fluorescence transfer between molecules of Akt labeled with CFP and YFP (Seveau et al. 2007. Cell. Microbiol. 9: 790). |
Practical C “Physical Study of the Interaction Between Aerolysin and the Plasma Membrane off Host Cells by Atomic Force Microscopy (AFM)”: This nanotechnology approach is designed to study in living cells several physical parameters at a nanometrical scale such as interaction forces between a ligand and its receptor, or the elasticity of membranes. Using functionalized cantilevers with the aerolysin protein from Aeromonas hydrophila, we will study the physical interaction of this bacterial effector with the plasma membrane of host cells. We will also study the potential uses of AFM as an imaging technique (Roduit et al. 2007. Biophys. J. In Press). |
Practical D “Analysis of the Interaction of Endocytic Compartments with the Salmonella enterica-Containing Vacuole using Correlative Light Electron Microscopy (CLEM)”: Electron microscopy is one of the most resolutive imaging techniques in biology, providing strong information about cell structure and morphology. CLEM in particular allows to detect by immunofluorescence particular cells that have followed a special treatment (transfection, specific labelling of a fluorescent tagged-protein or marker) and to analyze these particular cells by EM. We will study the internalization of rhodamine-gold in host cells and we will infect these cells with Salmonella enterica in order to visualize the interaction of the bacteria with different cellular compartments labelled by the fluorescent markers. Comparison with other pathogens will be also made. |
Practical E “Dynamic Imaging of the Secretion of Type III Effectors Using Multidimensional Time-Lapse Microscopy”: The use of a spinning disk coupled to a confocal microscope allows the faster acquisition of images from living cells minimizing the problems related to photo-bleaching and photo-toxicity. This approach will be used to study the use of the Type III Secretion System (TTSS) by Shigella flexneri during the invasion of target cells, and the cytoskeletal rearrangements associated with the secretion of the TTSS effectors. TTSS effectors tagged with a tetracysteine motif tag will be directly labelled with the fluorescein-based biarsenical dye (FLAsH). Use of 3D-reconstruction and deconvolution softwares will be coupled to the analysis (Enninga et al. 2005. Nat. Methods. 2: 959).
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Practical F “Multiparametric Analysis of Vacuoles Containing Salmonella enterica serovar Typhimurium using Fluorescent-Activated Cell Sorting (FACS): Flow cytometry is a powerful technique for analyzing large mixed populations of single cells or compartments. FACS is a more complex system, which not only quantifies the fluorescent signal but also separates the compartments from a mixed population that contains pre-selected characteristics such as fluorescence intensity, size and/or viability. This technique will be used to make an analysis of the maturation of the vacuole containing the bacterium Salmonella enterica at different times after infection (Steele-Mortimer et al. 1999. Cell. Microbiol. 1: 33). |