Imaging Noncanonical Autophagy and LC3-Associated Phagocytosis in Cultured Cells

Monitoring of ATG8 proteins by western blotting and immunofluorescence microscopy are the most common methods to monitor the autophagy pathway. However, it has recently been shown that ATG8 proteins can be lipidated to non-autophagosome, single-membrane compartments through a noncanonical autophagy pathway. This is commonly found to occur during macro-endocytic processes such as phagocytosis, where it has been termed LC3-associated phagocytosis, and upon lysosomotropic drug treatment. Therefore, care is required when interpreting data based on ATG8 in order to conclude whether a signal relates to the canonical or noncanonical pathway. Here we provide methods to monitor noncanonical autophagy through fluorescence microscopy.