LC3 forms functional nanoclusters on autophagosomes.

Autophagosome biogenesis depends on the coordinated action of proteins and lipids. However, how LC3B organizes at high density and contributes to autophagy-associated functions remains unclear. Using molecular dynamics simulations and super-resolution microscopy, we show that LC3B self-assembles into higher-order nanoclusters approximately 150 nm in size. These clusters form spatially distinct LC3B "islands" on the autophagosome membrane and show a preferential association with phosphatidylinositol-3-phosphate (PI3P) lipids. Molecular analysis of 296 structural clusters reveals a putative LC3B homo-clustering interface characterized by a distinct alternating hydrophobic-polar motif. To experimentally validate this interface, we generated four LC3B mutants targeting the clustering motif. STORM imaging of the LC3 mutants demonstrates a complete alteration in clustering dynamics, with diffuse, smaller LC3 clusters on the autophagosome. Further, motility analysis and interaction with the autophagic adaptor protein FYCO1 show abrogated autophagosome motility. Finally, loss of LC3B nanoclustering also significantly compromises autophagic entrapment of Streptococcus pneumoniae and results in elevated intracellular bacterial survival. Taken together, our results reveal LC3 clustering as a potential organizing principle that coordinates the spatial and temporal dynamics of autophagy.