A new cellular platform for studying autophagy

Atg8 proteins play a crucial role in autophagy. In yeast, there is a single Atg8 member, while mammals have six homologs categorized into LC3s and GABARAPs. The GABARAP subfamily consists of GABARAP, GABARAPL1, and GABARAPL2/GATE16 proteins, contributing to various autophagy stages. However, the intricacies among GABARAP proteins are complex and not well-understood. We introduce a new cellular platform to study autophagy, achieved by tagging GABARAP genes with N’-terminal 3xFlag epitopes fused to Clover or mScarletI fluorescent proteins using CRISPR/Cas9. This platform offers robust autophagy examination via flow cytometry, enabling real-time monitoring of GABARAP homologs at single-cell resolution through fluorescent microscopy. The 3xFlag epitope facilitates isolation and characterization of distinct autophagosomes labeled by specific GABARAP homologs. Our platform promises insights into the unique roles of GABARAP homologs in autophagy and possibly other cellular processes.