title: A proteolytic cleavage assay to monitor autophagy in Dictyostelium discoideum.
abstract: Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hinders the assessment of new mutants or drugs. One of these techniques, which has been used successfully in yeast and mammalian cells, but has not yet been described in Dictyostelium, is based on the presence of proteolytic fragments derived from autophagic degradation of expressed fusion proteins. Lysosomotropic agents such as NH 4Cl penetrate acidic compartments and raise their pH, thus allowing the accumulation and measurement of these cleaved fragments, which otherwise would be rapidly degraded. We have used this property to detect the presence of free GFP fragments derived from the fusion protein GFP-Tkt-1, a cytosolic marker. We demonstrate that this proteolytic event is dependent on autophagy and can be used to detect differences in the level of autophagic flux among different mutant strains. Moreover, treatment with NH4Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8.
authors.affiliation: Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Calvo-Garrido
authors.forename: Sergio
authors.initials: S
authors.lastname: Carilla-Latorre
authors.forename: Ana
authors.initials: A
authors.lastname: Mesquita
authors.forename: Ricardo
authors.initials: R
authors.lastname: Escalante
mesh_terms: D001343:Autophagy; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000166:cytology* / Q000378:metabolism*; D018613:Microscopy, Confocal; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D015415:Biomarkers; D011993:Recombinant Fusion Proteins