Submitted: 05/10/11

Dictyostelium discoideum is a good model of autophagy. However, the lack of autophagic flux techniques hin-ders 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 frag-ments derived from autophagic deg-radation of expressed fusion proteins. Lysosomotropic agents such as NH 4 Cl penetrate acidic compartments and raise their pH, thus allowing the accumula-tion 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 NH 4 Cl also facilitates the assessment of autophagic flux by confocal microscopy using the marker RFP-GFP-Atg8