Monitoring Autophagy in Dictyostelium

We describe here the use of confocal microscopy to detect the pattern of different autophagic markers and the differences expected in strains deficient in autophagy. Autophagy dysfunction might also lead to the formation of intracellular ubiquitinated protein aggregates that can be easily detected by immunofluorescence. In addition, we describe two different techniques that allow the assessment of the so-called autophagic flux, the progression of autophagosomes until their fusion with lysosomes. The first one is a proteolytic cleavage assay of cytosolic markers such as GFP-PgkA and the second the visualization of the RFP-GFP-Atg8 marker by confocal microscopy.
Source: Springer protocols feed by Microbiology - Category: Microbiology Source Type: news