LC3-positive structures are prominent in autophagy-deficient cells

Sci Rep. 2019 Jul 12;9(1):10147. doi: 10.1038/s41598-019-46657-z.


Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / physiology*
  • Autophagy-Related Protein 7 / genetics
  • Autophagy-Related Proteins / genetics
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • RNA-Binding Proteins / metabolism
  • Ubiquitin / metabolism
  • Vesicular Transport Proteins / genetics


  • Autophagy-Related Proteins
  • MAP1LC3A protein, human
  • MAP1LC3B protein, human
  • Microtubule-Associated Proteins
  • P62 protein, human
  • RNA-Binding Proteins
  • Ubiquitin
  • Vesicular Transport Proteins
  • ATG10 protein, human
  • ATG7 protein, human
  • Autophagy-Related Protein 7