Measurement of Bulk Autophagy by a Cargo Sequestration Assay.

The nonselective bulk sequestration of cytoplasm and its subsequent delivery to lysosomes for degradation was originally defined as autophagy or macroautophagy. However, both terms are now increasingly being applied in a generic sense to encompass the many recently described mechanisms for selective sequestration and degradation of individual cellular elements. We will therefore use the term bulk autophagy to denote the non-exclusive and largely nonselective process.

A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells.

Autophagy (self-eating) is a common term for various processes by which cellular components are transferred to lysosomes for degradation. In macroautophagy, intracellular membrane structures termed "phagophores" expand to encapsulate autophagic cargo into sealed, double-membrane vacuoles termed "autophagosomes," which subsequently may fuse with endosomes to form intermediary vacuoles called "amphisomes," and finally with lysosomes to have their contents degraded and recycled. Autophagy is frequently analyzed by monitoring phagophore- and autophagosome-associated markers such as LC3.

Autophagy of cytoplasmic bulk cargo does not require LC3.

To investigate the role of LC3 in bulk autophagy we compared its autophagic-lysosomal processing (using an improved quantitative immunoblotting method) with autophagic-lysosomal bulk cargo flux (measured by our established LDH [lactate dehydrogenase] sequestration assay) in amino acid-starved rat hepatocytes treated with cycloheximide to prevent new LC3 influx. Block-release experiments with the reversible autophagy inhibitors 3-methyladenine (3MA) and thapsigargin (TG) showed that while only 3MA suppressed phagophoric LC3 attachment (lipidation), both inhibitors prevented phagophore closure (cargo sequestration). Upon release from closure blockade, some autophagic-lysosomal LC3 flux was resumed even in the presence of 3MA, i.