Quantitative and temporal measurement of dynamic autophagy rates

Autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process that is dysregulated during many diseases. Established methods to quantify flux use steady state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 minutes, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannins inhibition of autophagy, which is further accelerated by rapamycin. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.