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.
Highlights
In yeast and mammalian cells, rapamycin (RAP) is often used as an autophagy activator, which acts by allosterically inhibiting the mechanistic target of rapamycin complex 1, which acts by allosterically inhibiting the mechanistic target of rapamycin complex 1, which directly phosphorylates Atg1 and connects nutrient availability to autophagy induction
Quantitative studies of the number of autophagic structures can be achieved by confocal microscopy of studies cells expressing a fusionof ofautophagic the green structures fluorescent can protein (GFP) with
Another kind of autophagic flux assay is based on the differential stability of green and red fluorescent fluorescent proteins proteins
Summary
Intracellular degradation processes are ubiquitous to all organisms and essential to maintain cellular homeostasis. A large number of proteins that participate in and regulate the autophagic process have been defined (termed Atg proteins) Many of these studies have been performed in model organisms, mainly yeast, as recognized recently by the. One of the organisms that has recently contributed to this area is Dictyostelium, a soil amoeba that was isolated and described by Kenneth Raper in 1935 Since it has become a well-established model in cell and developmental biology [11,12]. Since the first Dictyostelium autophagy mutants were described [29,32], it became clear that the severity of the phenotypes correlates with the level of autophagy inhibition [33] These phenotypes range from a complete lack of aggregation to incomplete development, characterized by the formation of multi-tipped structures and aberrant fruiting bodies, which has facilitated the isolation and characterization of many autophagy mutants (Figure 1B). The formation of multipleoftips is characteristic latter, the formation multiple tips is characteristic some of the strains.of some of the strains
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