Quantification of Autophagosome Size and Formation Rate by Electron and Fluorescence Microscopy in Baker’s Yeast

The use of both transmission electron microscopy and fluorescence microscopy have provided tremendous advances to our understanding of autophagosome formation in baker’s yeast, Saccharomyces cerevisiae. In the last decade, parallel techniques have been developed for both types of microscopy that allow the quantification of the rate of autophagosome formation. Importantly, these techniques, unlike other measures of total autophagic flux, allow a researcher to distinguish between effects on autophagosome size and autophagosome number. This has led to the discovery that certain autophagy proteins (e.g., Atg8) contribute primarily to the control of autophagosome size, whereas others (e.g., Atg9) are principally involved in controlling autophagosome number, suggesting different roles for these proteins in the autophagosome formation process.In this chapter, we present two methods for quantifying autophagosome formation in yeast. One, based on electron microscopy analysis of autophagic bodies in the vacuole, can give estimates of both autophagosome size and number. The other, based on live-cell imaging of growing autophagosomes labeled with GFP-Atg8, can provide information on the rate of autophagosome formation. Together they provide a robust toolbox for analyzing the roles of different proteins in the process of autophagosome formation.