How and why to study autophagy in Drosophila: it's more than just a garbage chute.
Bottom Line: This way, autophagy contributes to the homeodynamic turnover of proteins, lipids, nucleic acids, glycogen, and even whole organelles.Autophagic activity is increased by adverse conditions such as nutrient limitation, growth factor withdrawal and oxidative stress, and it generally protects cells and organisms to promote their survival.Here we discuss the different microscopy-based, biochemical and genetic methods currently available to study autophagy in various tissues of the popular model Drosophila.
Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.Show MeSH
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Mentions: Autophagic structures have certain unique features that make it possible to reliably recognize them. Depending on the sample preparation technique and type of fixation used, the closely apposed membranes of both phagophore cisterns and autophagosomes may open up, and thus a characteristic cleft appears between the two membrane sheets (as seen in Fig. 2A,C and E; note that such vesicles are very rarely observed in neurons of wild type adult flies, shown in Fig. 2B). This phenomenon is often seen in samples prepared by chemical fixation of cells and tissues by a glutaraldehyde-containing isoosmotic solution, which is followed by dehydration of fixed samples and embedding into a resin [6,10,13,21–23]. Intracellular structures likely undergo a variable extent of shrinkage during fixation and embedding according to local conditions in the surrounding cytoplasm, which is why this cleft may be seen only in a subset of autophagic structures. The appearance of this cleft is unlikely to be specific for different cell types or organisms, as it has been documented elsewhere as well [9,24]. The membranes of these early autophagic structures are of the thin type, based on which autophagosomes can be distinguished from interdigitations observed between neighboring cells in some tissues, as the membrane of these are of the thick type (plasma membrane). Autophagosomes contain undigested material, the morphology of which appears very similar to the surrounding cytoplasm (Fig. 2A and C). In contrast, the content of autolysosomes is mostly heterogeneous, as it shows morphological signs of ongoing degradation ranging from recognizable to finally unidentifiable cytoplasmic material like mitochondria, RER, ribosomes and so on. The heterogeneous morphology can be the result of multiple subsequent fusion events resulting in a multifocal appearance. Both primary lysosomes and actively digesting autolysosomes and endolysosomes contain acid phosphatase, which can be detected in the electron microscope using a classical, very simple enzymatic reaction [25,26]. In addition, immunogold labeling for lysosomal proteins using antibodies (or tagged reporters and anti-tag antibodies) to cathepsin proteases or lysosomal membrane proteins can also be used to identify lysosomes in ultrastructural studies .
Affiliation: Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Pázmány s. 1/C. 6.520, Budapest H-1117, Hungary.