Macroautophagy is characterized by the sequestration of cytoplasmic material within
autophagosomes for bulk degradation by lysosomes. Autophagosomes, by definition, are twomembraned
and contain degenerating cytoplasmic organelles or cytosol,35,36 which allows
them to be distinguished by transmission electron microscopy from other types of vesicles such
as endosomes, lysosomes or apoptotic blebs.3 The fusion between autophagosomes and
lysosomes generates autolysosomes, in which both the autophagosome inner membrane and
its luminal content are degraded by acidic lysosomal hydrolases. This catabolic process marks
the completion of the autophagic pathway. When the fusion of autophagosomes with lysosomes
is blocked, the former accumulate in spite of autophagy inhibition.36,37 Hence, a massive
increase in the number of autophagosomes is by no means a demonstration that the autophagic
pathway is induced, and functional tests are required to investigate autophagy. A very
comprehensive description of the assays for monitoring autophagy in higher eukaryotes and a
set of guidelines for their interpretation has been recently provided by Klionsky et al.38 One
technique commonly employed to detect autophagy relies on the redistribution of GFP-LC3
fusion proteins into vesicular structures (which can be autophagosomes or autolysosomes).3,
39 However, the exclusive use of GFP-LC3 as a marker of autophagy is not sufficient to
diagnose an enhanced autophagic catabolism.38