Electron Microscopic Analyses of Autophagy-Related Organelles in Mammalian Cells

Satoshi Waguri, Takefumi Uemura, Masaya Yamamoto and Atsuko Yabashi

Abstract: Autophagy is involved in the intracellular bulk degradation system, and is crucial for cellular homeostasis and stress response mechanisms. Upon autophagy signal, isolation membrane (phagophore) emerges and sequestrates the cytoplasm, which is closed to become autophagosome. The constituents are then degraded after fusing with lysosomes. Because this system involves dynamic changes in the membranous structures, analyses at electron microscopic levels are required to understand the detailed processes. However, some precautions are necessary for appropriate evaluation of the autophagy-related structures.  Especially, preservation of the double membrane structure of the isolation membrane is greatly influenced by fixation and other sample preparation procedures. In this review, we first describe how we should identify and evaluate several autophagy-related organelles while pointing out some precautions, and second, introduce recent understanding on origins of the isolation membrane, a long-standing issue in autophagy research.

Key words: autophagy, isolation membrane, phagophore, electron microscopy, fixation methods