Dynamics of Atg5–Atg12–Atg16L1 Aggregation and Deaggregation

Macroautophagy is a physiological process that is implicated in various pathological conditions, including neurodegenerative diseases and cancer. The execution of canonical autophagy is regulated by a core signaling cascade and it involves two well-characterized, ubiquitin-like conjugation systems—the Atg5/Atg12/Atg16L1 and the Atg8-phosphatidyl ethanolamine (PE), which are both catalyzed by Atg7. The conjugation of Atg5–Atg12 and the subsequent interaction with the positive regulator Atg16L1 are essential for the conjugation of Atg8 to PE and the subsequent formation of autophagosomes. The interaction between Atg5–Atg12 complex and Atg16L1 is highly dynamic, induced upon activation of the autophagic process, and required for the recruitment of the At5–Atg12 complex to sites of autophagosome formation. Monitoring the Atg5–Atg12–Atg16L1 aggregation and deaggregation may be used not only as means to study the dynamics of autophagy, but in another important point, it may provide important insights on the basic molecular mechanisms of autophagy in physiological and pathological settings. In this chapter, we describe methods of monitoring the Atg5–Atg12–Atg16L1 aggregation and deaggregation, with emphasis on prostate cancer.