Isolation of autophagosome subpopulations after induction of autophagy by calcium.

Autophagy is a dynamic process accomplished by the generation and maturation of autophagosomes. Isolation of autophagosomes and subsequent compositional analysis can provide information about their biogenesis mechanism. In this article, HEK293 cells expressing GFP-LC3 were treated by calcium phosphate precipitates (CPP) to induce autophagy. The autophaogomes induced by CPP were tubular and vesicular structures, extensively formed in the cytosol. After all membranes in the cell lysate were fractionated by differential centrifugation, autophagosomes from light and heavy membranes were isolated by immuno-precipitation, using antibodies against GFP-LC3 and Atg5. We found that GFP-LC3 and Atg5 positive autophagosomes represented distinctive subpopulations. Judged from the molecular markers associated, including organelle markers and Atg proteins, GFP-LC3 positive autophagosomes were overall at the later biogenetic stage. Furthermore, both GFP-LC3 and Atg5 positive autophagosomes from light membranes were less mature than those from heavy membranes. We have established a method to isolate subpopulations of autophagsomes for further characterization.