Monitoring Autophagic Flux in the Model Single-Celled Microalga Chlamydomonas reinhardtii.

ABSTRACT

Autophagy is a catabolic process by which eukaryotic cells degrade and recycle unnecessary or damaged intracellular components to maintain cellular homeostasis and to cope with stress. The development of specific tools to monitor autophagy in microalgae and plants has been fundamental to investigate this catabolic pathway in photosynthetic organisms. The protein ATG8 is a widely used molecular marker of autophagy in all eukaryotes, including the model microalga Chlamydomonas reinhardtii. The drug concanamycin A, a specific inhibitor of vacuolar ATPase, has also been extensively used to block autophagic flux in the green lineage. In Chlamydomonas, inhibition of autophagic flux by concanamycin A has been shown to prevent the degradation of ribosomal proteins and the formation of lipid bodies under nitrogen or phosphorous starvation. Here, we detail how the abundance and lipidation state of ATG8 can be used to monitor autophagic flux in Chlamydomonas by western blot analysis.