Analysis of autophagosome membrane cycling by fluorescence microscopy

Autophagy is a physiological process functionally linked to cellular dynamics during starvation, cardiomyopathies, neurodegeneration, cellular immunity, and certain cancers. Although nearly 30 autophagy-related (ATG) genes have been identified and characterized, the molecular mechanisms of this process are only partially understood. One aspect of the pathway that has been intensely studied is the identity of the membrane source for newly formed autophagosomes. Although it occurs at a basal level, autophagy is an inducible process. The process of autophagosome formation involves recruitment and delivery of membrane and recycling of Atg proteins. Despite continuing attempts to identify the source of the autophagosome membrane, we are only recently beginning to understand the nature of autophagosome formation and the role of membrane protein cycling in this process. There now exists an assay utilizing fluorescence microscopy to monitor the localization, and therefore the movement, of membrane-associated Atg proteins. We describe here a method that allows visualization of Atg membrane proteins in order to observe their potential source membranes and also to determine the temporal order of action of other Atg proteins with regard to their movement.