Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor

Mitochondrial dysfunction is implicated in many human diseases and occurs in normal aging. Mitochondrial health is maintained through organelle biogenesis and repair or turnover of existing mitochondria. Mitochondrial turnover is principally mediated by mitophagy, the trafficking of damaged mitochondria to lysosomes via macroautophagy (autophagy). Mitophagy requires autophagy, but is itself a selective process that relies on specific autophagy-targeting mechanisms and thus can be dissociated from autophagy under certain circumstances. Therefore, it is important to assess autophagy and mitophagy together and separately. We sought to develop a robust, high-throughput, quantitative method for monitoring both processes in parallel. Here we report a flow cytometry-based assay capable of rapid parallel measurements of mitophagy and autophagy in mammalian cells using a single fluorescent protein biosensor. We demonstrate the ability of the assay to quantify Parkin-dependent selective mitophagy in CCCP-treated HeLa cells. In addition, we show the utility of the assay for measuring mitophagy in other cell lines, as well as for Parkin-independent mitophagy stimulated by deferiprone. The assay makes rapid measurements (10,000 cells per 6 seconds) and can be combined with other fluorescent indicators to monitor distinct cell populations, enabling design of high-throughput screening experiments to identify novel regulators of mitophagy in mammalian cells.