ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.

Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.