title: DictyosteliumCultivation, Transfection, Microscopy and Fractionation.
abstract: The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to theDictyostelium discoideumprotein TSPOON that is part of an ancient heterohexamer membrane trafficking complex (Hirstet al., 2013). TSPOON is the product of thetstDgene inDictyosteliumand is not required for growth or the developmental cycle in this organism.Dictyosteliumamoebae will exist in a vegetative phase where growth is sustained by the phagocytosis of bacteria. When this food source is spent they enter a developmental phase where the amoebae aggregate, via chemotaxis to extracellular waves of cAMP, into multicellular structures that subsequently form a fruiting body containing viable spores (Muller-Taubenbergeret al., 2013). In the laboratory this cycle takes less than 24 h to complete and as a further aid to manipulation the requirement for a bacterial food source has been circumvented by the derivatisation of the wild type and isolation of axenic strains that can also grow in a nutrient rich broth. Axenic strains like Ax2 are the mainstay of laboratory research usingDictyostelium(Muller-Taubenbergeret al., 2013). A description ofDictyosteliumcell cultivation, the generation of cell lines that overexpress TSPOON-GFP and TSPOON null cells, and subsequent analysis (Muller-Taubenberger and Ishikawa-Ankerhold, 2013) is detailed below.
authors.affiliation: Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hirst
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Kay
authors.affiliation: MRC Laboratory of Molecular Biology, Cambridge, UK.
authors.forename: David
authors.initials: D
authors.lastname: Traynor