Table 1.

Assays for autophagosome maturation and autophagic degradation^a

Assay	Description	Read out
Protease sensitivity of LC3-II	LC3-II is degraded by lysosomal proteases following fusion of the autophagosome with the lysosome (53). Lysosomal protease inhibitors, which inhibit LC3-II degradation by the autolysosome, increase the steady-state level of LC3 II (53, 56).	Protein degradation by the autophagic pathway
p62 Degradation	The p62/SQSTM1 protein directly binds LC3-II on the autophagosome membrane (54). p62 is degraded within the autolysosomes (55). A decrease in the steady state level of p62 following induction of autophagy indicates successful protein degradation through the pathway (55, 56).	Autolysosome formation
LC3-II-lysosome colocalization	The cellular locale of autophagosome-associated GFP-tagged LC3-II can be monitored by fluorescence microscopy (67). During the initial stages of the autophagic pathway, colocalization of LC3-II with lysosomal markers is low. As autophagosomes mature and fuse with lysosomes, colocalization with lysosomal markers increases. Cellular lysosomes can be visualized by staining for protein markers such as LAMP-2, LAMP-1, and cathepsin D (31).	Autolysosome formation
Tandem-tagged GFP-RFP-LC3	Tandem-tagged RFP-GFP-LC3 localizes to the autophagosome membrane following induction of autophagy (58). Only the signal generated by the GFP protein is sensitive to the acidic and/or proteolytic conditions in the lumen of mature autophagosome and lysosomes. Colocalization of GFP and RFP signals is observed on early or immature autophagosomes. As autophagosomes mature, the GFP signal is lost, leading to only RFP fluorescence.	Autophagosome maturation
Transmission electron microscopy	Autophagosomes can by identified by TEM as membrane-bound structures containing cytoplasmic material. Immature autophagosomes (AVi) show a double membrane visible as two membrane bilayers separated by an electron-lucent cleft. These vacuoles contain cytosol and/or organelles that appear morphologically intact (56). Mature or degradative vesicles (AVd) typically show partial degradation of the enclosed cytoplasmic material, as well as increased electron density in the lumen of the vesicle.	Autophagosome maturation

^aAn extensive discussion of known assays for analysis of autophagic signaling, autophagosome formation, and all other aspects of the pathway can be found in reference 53. TEM, transmission electron microscopy.