Estimating the size and number of autophagic bodies by electron microscopy

Abstract

Much 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 ume6∆ strain.

Keywords: autophagosome; autophagy; electron microscopy; stereology; vacuole; yeast.

Figure 1. Example of autophagic body outlines derived from an EM image.

Figure 2. Example of manually fitting the graph of g(y) to the actual data.

Figure 3. Effect of sample size on the results obtained by these methods. (A) Variation in estimated mean radius due to sample size. For each sample size, 10 random subsamples of that size were taken from a given sample of 1600 measurements and used to estimate the mean radius. The percent difference between this estimate and the reference estimate (obtained using the entire 1600 measurements) is plotted. (B) Variation in estimated body number due to sample size. A simulation method including a clumping routine (File S6; mu = 5, sigma = 0.4, balls = 80, recognition threshold = 30) was used to generate a set of simulated cross sections for a varying number of simulated cells. The percent difference between the number of cross sections per cell predicted by each run of the simulation and the reference number (the average of the 1000-cell simulations) is plotted.

Figure 4. Analysis of autophagosome size and number in ume6∆ cells. (A) Pho8∆60 assay of autophagic flux during nitrogen starvation in SEY6210 pho13∆ pho8∆60 (WT), and ume6∆ cells. (B) Autophagic bodies in SEY6210 pep4∆ vps4∆ (WT) and ume6∆ cells after 2 h of nitrogen starvation. Scale bars: 500 nm. (C) Results from the analysis of the autophagic bodies following the protocol described in this paper. N > 200 cells, and > 1000 bodies. SD, standard deviation. (A and B) were adapted from reference (copyright National Academy of Sciences [USA]).