Abstract
Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable.
Methods:
In this study, we developed a small-molecule fluorogenic probe (
AFG-1
) that facilitates real-time imaging of autophagic flux in both intact cells and live mice.
AFG-1
is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step,
AFG-1
responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated.
Results:
This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO
-
and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore,
AFG-1
has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness.
Conclusion:
Due to the specificity, easy operation, and the dynamic information yielded,
AFG-1
should serve as a potential tool to explore the roles of autophagy under various pathological settings.