Methods to Monitor and Quantify Autophagy in the Social Amoeba Dictyostelium discoideum

Abstract

Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages from initiation to final degradation of cargo are finely regulated. Therefore, the study of this process requires the use of a palette of techniques, which are continuously evolving and whose interpretation is not trivial. Here, we present the social amoeba Dictyostelium discoideum as a relevant model to study autophagy. Several methods have been developed based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophagy genes and proteins, and examination of the autophagic flux with various techniques. In this review, we discuss the pros and cons of the currently available techniques to assess autophagy in this organism.

Keywords: Dictyostelium; autophagic markers; autophagy; cleavage assays; flux assays.

Figure 1

Dictyostelium as a model for autophagy. Dictyostelium discoideum is a social amoeba that enters a developmental program during starvation. (A) Scheme of the Dictyostelium developmental cycle. Individual amoebas aggregate to form mounds of cells that undergo different stages of development that culminate with the formation of a fruiting body containing spores. (B) The lack of autophagy in Dictyostelium leads to developmental arrest either at the aggregation stage or at the mound stage. In the latter, the formation of multiple tips is characteristic of some of the strains.

Figure 2

A short-term rapamycin (RAP) treatment does not induce autophagic flux in Dictyostelium. Dictyostelium Ax2(Ka) cells expressing green fluorescent protein (GFP)-Atg8 were treated or mock-treated with RAP at 500 nM for 2 h. One hour before the end of the treatment, cells were incubated or not with a protease inhibitor cocktail (PI, Roche 11873580001) at 2.5×. (A) Representative maximum projections of live cells under the treatments described above. Scale bars, 10 µm; (B) Median and interquartile ranges of the number of GFP-Atg8 structures per cell during the mentioned treatments. Each dot represents one cell; 162–168 cells per condition were counted. The values of λ that define the Poisson distribution of each data set and differences between them were calculated as described before [34] (**** p ≤ 0.0001; ns, p > 0.05). No significant differences were observed by quantification of the percentage of cells with GFP-Atg8 dots under RAP treatment (not shown).

Figure 3

Visualization of autophagosomes in Dictyostelium by TEM. Electron micrographs of a Dictyostelium Ax2(Ka) cell treated with the autophagy inducer drug AR-12 at 2.5 µM for 2 h and with PI at 2.5× for 1 h. For imaging, cells were fixed for 1 h with 2% glutaraldehyde and stained for 30 min with a 2% osmium/0.1 M imidazole solution. Fixed cells were pelleted, washed in phosphate-buffered saline (PBS), and further processed, as previously described [38]. The red arrow marks the enlarged panel showing two autophagosomes; the green arrow marks the enlargement where a fusion event between a late endosome and an autophagosome can be observed. Scale bar, 2 µm.

Figure 4

Atg18 and Atg8 as autophagic markers. (A) Atg18 is necessary for autophagosome formation, but it is released from mature autophagosomes. The cytosolic Atg8 protein is conjugated to phosphatidylethanolamine (PE) upon induction of autophagy, and is stably integrated into both the outer and the inner membranes of the phagophore. Therefore, the Atg8 fraction residing inside the autophagosome is degraded, together with other autophagic cargos, after lysosomal fusion; (B) red fluorescent protein (RFP)-GFP-Atg8 binds the autophagosomal membranes when autophagy is induced. Inside the autolysosomes, GFP is quenched due to the low pH, but RFP remains fluorescent. The increase in yellow and red Atg8 indicates induction of the autophagic flux, while the increase only in yellow Atg8 structures implies autophagic flux blockage.

Figure 5

Protein cleavage assay. AX4 wild-type (WT), atg1^-, or atg7^- cells constitutively expressing GFP-PgkA (phosphoglycerate kinase) were subjected to two 150 mM pulses of NH4Cl, each of 2 h, as previously described (see text). Total cell extracts were analyzed by western blot using an anti-GFP antibody. Free GFP cannot be detected in autophagy-deficient strains.

Figure 6

Wild-type (WT) (AX4) and atg1^- cells expressing the marker RFP-GFP-Atg8 were incubated for four hours with two pulses of 100 mM NH₄Cl and visualized by confocal microscopy. The arrows show the presence of red-only puncta in WT that presumably represent autophagolysosomes. Cells lacking Atg1 typically show a large aggregate and no red-only puncta. Scale bar 5 μm.

Figure 7

Detection of ubiquitinated protein aggregates by confocal microscopy. WT (AX4) and vmp1⁻ cells were fixed and prepared for immunocytochemistry detection of ubiquitin (red), and analyzed by confocal microscopy. Autophagy-deficient vmp1⁻ cells are not able to degrade proteins by autophagy and accumulate large ubiquitinated aggregates (denoted by white arrows). Scale bars 5 μm.