Assays to monitor autophagy in Drosophila

Abstract

The term autophagy refers to the engulfment and degradation of cytoplasmic components within the lysosome. This process can benefit cells and organisms by removing damaged, superfluous, or harmful cellular components, and by generating a supply of recycled macromolecules that can support biosynthesis or energy production. Recent interest in autophagy has been driven by its potential role in several disease-related phenomena including neurodegeneration, cancer, immunity and aging. Drosophila provides a valuable animal model context for these studies, and work in this system has also begun to identify novel developmental and physiological roles of autophagy. Here, we provide an overview of methods for monitoring autophagy in Drosophila, with a special emphasis on the larval fat body. These methods can be used to investigate whether observed vesicles are of autophagic origin, to determine a relative rate of autophagic degradation, and to identify specific step(s) in the autophagic process in which a given gene functions.

Coordination of autophagosome–lysosome fusion and transport by a Klp98A–Rab14 complex in Drosophila

“…Vesicles labeled with these markers showed almost no overlap in Rab14-depleted cells, with a correlation coefficient similar to the effects of Klp98A depletion ( Figs 3B, 5E,F). We further examined autophagosome-lysosome fusion in Rab14-depleted cells using the acidic compartment dye LysoTracker Red, which in fat body cells specifically labels autophagy-induced acidic vesicles (Mauvezin et al, 2014;Scott et al, 2004). Consistent with a defect in autolysosome formation, strong punctate LysoTracker Red labeling was observed in starved control cells but not in Rab14-depleted cells (Fig.…”

“…1A). To study the function of Klp98A in autophagy, we examined the effects of Klp98A loss-and gain-of-function on mCherry-tagged Atg8a protein (mCh-Atg8a), a well-characterized marker of autophagosomes and autolysosomes (Klionsky et al, 2012;Mauvezin et al, 2014). Klp98A depletion in fat body cells led to a reduction in the overall number of vesicles marked by mCh-Atg8a, and to their redistribution towards the perinuclear region of the cell ( Fig.…”

Mitf is a master regulator of the v-ATPase forming an Mitf/v-ATPase/TORC1 control module for cellular homeostasis

“…S3B). Finally, we turned to a well-characterized physiological setting for TORC1 modulation, specifically starvation-induced inactivation of TORC1 in fly salivary glands cells (Mauvezin et al, 2014). In this background, we also detected a shift of Mitf to the nucleus (supplementary material Fig.…”

Autophagosomes take the Klp98-A train

“…We found that Klp98A gain-or loss-of-function modified the localization of vesicles marked by the autophagosomal membrane protein Atg8/LC3 and of lysosomes marked by LAMP1. 13 Klp98A depletion significantly disrupted the localization of the 3 populations of vesicles involved in autophagy, i.e., autophagosomes, lysosomes, and autolysosomes. Each of these vesicles was aberrantly localized to the perinuclear area upon Klp98A depletion, and to the cortical region of cells overexpressing Klp98A.…”