(635d) A New Methodology for Quantitative and Temporal Measurement of Autophagy.

Autophagy is a multi-step intracellular process by which cells recycle their misfolded proteins and damaged organelles into primary building blocks. The cellular materials are sequestered by double-membrane vesicles called autophagosomes, which fuse with lysosomes to form autolysosomes, which then degrade the sequestered material into fatty acids and amino acids. Autophagy is linked to many diseases, including cancer, neurodegenerative diseases, and infectious diseases. The significance of autophagy in various cellular processes is growing, yet sensitive and temporal quantification of the dynamic autophagic process remains a challenge. Quantifying autophagy temporally allows the fundamental understanding of autophagy and will be useful for biomedical and biotechnology applications. However, many current methods lack the temporal quantification making them inadequate to characterize the dynamic nature of autophagy.

We developed a new single-cell fluorescence microscopy-based methodology to measure all the steps involved in autophagy temporally and quantitatively. The methodology considers the whole autophagic process as a multistep process governed by the rate of formation of autophagosomes (R1), the rate of fusion of autophagosomes with lysosomes (or rate of formation of autolysosomes) (R2), and the rate of degradation of autolysosomes(R3). All three rates can be measured temporally using an instantaneous rate approach, thus characterizing the change in the functional state of the system over time. This methodology would allow systematic characterization and kinetic analysis of autophagy under non-steady state conditions. Therefore, the methodology can be used to study various external perturbations such as small molecules drugs and dynamic perturbations such as viruses. Additionally, the methodology would further assist in the development of new therapies to regulate autophagy accurately.

We validated the new methodology using rapamycin, a well-studied small molecule drug commonly used to regulate autophagy. A tandem green and red fluorescent clonal human cell line was used to monitor autophagosomes and autolysosomes in real-time. We measured the change in the rates of the autophagic steps temporally during rapamycin treatment using the new methodology through direct quantification of autophagosomes and autolysosomes as a function of time. The measured autophagic rates not only provides insights into the effect of rapamycin on autophagy but also the cellular response to the treatment. Furthermore, we are developing a mathematical model that characterizes the autophagic pathway in response to rapamycin treatment based on temporal change in the autophagosomes and autolysosomes data. The predicted autophagic rates from the mathematical model will then be compared to the measured autophagic rates based on methodology indicating the accuracy of the model as well as the methodology. Additionally, the mathematical model characterizes the concentration-dependent effect of rapamycin on the autophagic pathway and can be easily extended to other rapalogs (drugs analogs to rapamycin).

In conclusion, this methodology will lay the foundation for temporal quantification and kinetic analysis of the autophagic system. As a future direction, we will be applying this methodology to study the effect of the various small-molecule drugs as well as viruses such as dengue and Zika virus on autophagy.