Quantitative super-resolution microscopy reveals distinct ULK1 oligomeric states and nanoscopic morphologies during autophagy initiation

Autophagy is an evolutionarily conserved process for degradation and recycling of intracellular components. Although autophagy has been extensively studied over the past decades, it still remains unclear how autophagosome formation occurs in response to starvation. Here, using quantitative Photoactivated Localization Microscopy (qPALM) we have analyzed the nanoscopic spatial distribution and the oligomeric states of endogenous ULK1, the central autophagy induction regulator. For this, we introduced the gene of mEos2, a photoswitchable fluorescent protein, into the ULK1 allele of the HeLa cell genome using a genome-editing technique to express mEos2-ULK1 at an endogenous level. With single-molecule sensitivity, we found that ULK1 exists as various oligomers with clustered and arc-shaped structures throughout the cytoplasm. Upon induction of autophagy by amino acid starvation, a small fraction of ULK1 molecules formed higher-order oligomers localized to larger spherical structures with radii up to 380 nm. Cross-correlation analysis of two-color PALM data of ULK1 and its interaction partner Atg13 revealed that ULK1 structures composed of more than ~40 molecules form under starvation only when ULK1 and Atg13 are colocalized. Through two-color PALM analysis of ULK1 and the endoplasmic reticulum (ER) compartment, we found that these autophagic ULK1 structures occur in a close proximity of the ER within 800 nm. These results indicate that ULK1 might engage in the initiation of autophagosome formation at and near the ER where its cluster size expands to contain more than 40 ULK1 molecules. This quantitative analysis of endogenous ULK1 has provided unprecedented insights into the origin of the autophagy initiation structures.