LC3 forms functional nanocluster on autophagosome

Autophagosome biogenesis relies on the intricate coordination of proteins and lipids, with LC3B proteins persistently anchored to the double-membraned autophagosomes through a lipid anchor. However, little is known about how LC3B is organized in high concentration, its spatial distribution, and the mechanisms underlying its protein-mediated tethering on membranes. Using molecular dynamics simulations and super-resolution microscopy, we demonstrate that LC3B self-assembles to form higher-order clusters, averaging 150 nm in size. Interestingly, simulations provided cue for LC3B and phosphatidylinositol lipid specificity, and STORM imaging confirmed a clear overlap of LC3B-enriched regions with phosphatidylinositol-3-phosphate lipids. Together, LC3B nanoclusters on these lipids form spatially distinct “islands” on the autophagosome. Additionally, molecular analysis of 296 clusters revealed that clustering is driven by a unique rear binding pocket in Loop6 defined by alternating hydrophobic and polar residues. We generated four mutants to disrupt the characteristic self-assembly motif, with all four mutants resulting in aberrant cluster formation and impaired autophagosome motility. These findings highlight that LC3B self-assembly is crucial for autophagy and suggest a spatiotemporal mechanism regulating LC3B function.