Estimating the size and number of autophagic bodies by electron microscopy

Abstract

ProtocoL ProtocoL M uch recent and ongoing research is focused on understanding the mechanisms and regulation of autophagy, a cellular self-degradation pathway with many links to human health. Although many assays exist to measure the total magnitude of autophagy, electron microscopy remains the tool of choice for the determination of the size and the number of autophagosomes formed in a given mutant or under given induction conditions. Here we present a detailed protocol for measuring autophagic bodies in the yeast Saccharomyces cerevisiae by electron microscopy. Furthermore, we present an improved mathematical method for estimating body size and a new method for estimating body number. Finally, we include a discussion of the merits and limitations of these methods and an example of their application to autophagic bodies formed in the ume6Δ strain.

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

“…3A); the size and number of autophagic bodies were quantified and estimated as described previously. 21 The average size of the autophagic bodies in both mutants was not significantly different compared to the WT (Fig. S2).…”

“…Finally, our data suggest that the S122D mutation results in an enhanced interaction in particular between Atg9 and Atg27, one of the components required for efficient anterograde trafficking of Atg9. 21,45 We note, however, that Atg23 and Atg27 do not appear to be conserved in higher eukaryotes, so different components my take the place of these proteins if this regulatory aspect of Atg9 trafficking is conserved beyond fungi.…”

“…21,49,57,58 Antibodies against Atg9, Ape1 and Pgk1 (a generous gift from Dr. Jeremy Thorner, University of California, Berkeley), and a commercial antibody that reacts with protein A (no longer available) were used as described previously. 15,35 Anti-Dpm1 was from Molecular Probes/Invitrogen (A-6429).…”

“…18 Thus, Atg9 may move between these peripheral sites and the PAS, and earlier studies have suggested that this cycling of Atg9 is required for autophagosome formation, functioning in some manner to direct membrane to the expanding phagophore. 16,[19][20][21] In mammalian cells, ATG9 also displays dynamic cycling, moving between the trans-Golgi network and endosomes in response to nutritional changes. A subpopulation of ATG9 colocalizes with both LC3, a mammalian homolog of Atg8, and RAB7, a late endosomal protein.…”

Measurement of radiation-induced autophagic flux in human brain cancer cells

“…The main function of autophagy is to recycle damaged or old cellular constituents by encapsulating cargo within autophagosomes that are then trafficked to fuse with lysosomes, allowing lysosomal hydrolytic enzymes to catabolize the cellular material into simple macromolecular subunits. Induction of autophagy above basal levels provided some of the first insights into the complex role of this dynamic process in cell death and its role in radiation sensitization [4,5].To provide a proof-of-principle, morphomics data was collected and analyzed for human glioblastoma (U87) monolayer cultured cells irradiated with 0 or 10 Gy  rays [2] and 3 days after treatment were collected and fixed for TEM (the most sensitive method to monitor autophagy [6]). Biased sampling methods were used to selectively acquire at least 30 cell profiles for each treatment.…”