Measuring Autophagosome Flux

Abstract

Autophagy is an evolutionarily conserved catabolic process that plays an import role in cellular proteostasis. The continual degradation, and recycling, of portions of the cytoplasm through autophagy eliminates unused and toxic proteins and organelles, promoting a functional proteome and cellular function. Autophagy also serves as an important adaptive mechanism that protects against metabolic perturbation. Loss of autophagy activity has major detrimental effects and has been shown to lead to neuronal proteotoxicity, protein aggregation, and cell death onset associated with neurodegeneration. Studies aimed at modulating autophagy activity have shown promising results in clearing toxic protein cargo and preserving neuronal viability. Neurons are characterized by a particularly efficient autophagy system. However, to finely control autophagy activity requires the precise and accurate measurement of the autophagy flux, i.e., the rate of flow along the entire autophagy pathway. Fluorescence microscopy has substantially contributed to the assessment of autophagy, due to its ability to identify the autophagy pathway intermediates, and to describe them kinetically, in the entire cell volume. However, the quantitative discernment between autophagy pathway intermediates, particularly the autophagosome pool size and the autophagosome flux, has remained challenging. Here, we describe a single-cell analysis approach that allows the characterization of the autophagy system in terms of the pathway intermediate steady-state pool size, the autophagosome flux, and the transition time.