ABSTRACT: Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway. Based on the theoretical framework of metabolic control analysis, where the autophagosome flux is the quantitative description of the rate a flow along a pathway, here we treat the autophagy system as a multi-step pathway. We describe a single-cell fluorescence live-cell imaging-based approach that allows the autophagosome flux to be accurately measured. This method characterizes autophagy in terms of its complete autophagosome and autolysosome pool size, the autophagosome flux, J, and the transition time, ?, for autophagosomes and autolysosomes at steady state. This approach provides a sensitive quantitative method to measure autophagosome flux, pool sizes and transition time in cells and tissues of clinical relevance. ABBREVIATIONS:ATG5/APG5, autophagy-related 5; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; J, flux; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin kinase; nA, number of autophagosomes; nAL, number of autolysosomes; nL, number of lysosomes; p-MTOR, phosphorylated mechanistic target of rapamycin kinase; RFP, red fluorescent protein; siRNA, small interfering RNA; ?, transition time; TEM, transmission electron microscopy.
