USING PHOTOACTIVATABLE PROTEINS TO MONITOR AUTOPHAGOSOME LIFETIME

Many conditions are now known to cause autophagosome proliferation in cells and organisms including amino acid and serum starvation, ER and oxidative stress, and pathogen infection. Autophagosome proliferation is also observed in disease states and developmental programs. The widespread use of GFP-Atg8 fusion molecules has provided a simple way to visualize the proliferation of autophagosomes in cells. However, GFP-Atg8 markers do not reveal the underlying cause of autophagosome proliferation. Two processes regulate the number of autophagosomes present in cells: (1) formation of the structures and (2) their turnover through fusion with lysosomes. Here we describe the use of photoactivatable proteins to decouple the processes of autophagosome formation from autophagosome turnover. Photoactivatable proteins fused to Atg8 homologs make it possible to pulse-label existing populations of autophagosomes in living cells. The fate of those pulse-labeled autophagosomes can then be monitored to determine autophagosome lifetime. This assay is applicable to both engineered tissue culture models and transgenic organisms expressing photoactivatable proteins fused to Atg8 homologs.