A tunable fluorescent timer method for imaging spatial-temporal protein dynamics using light-driven photoconvertible...

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial-temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation. Herein we propose a light-driven timer concept that could report relative protein ages at specific sub-cellular locations, by weakly but chronically illuminating photoconvertible fluorescent proteins inside cells. This new method exploits light, instead of oxygen, as the driving force. Therefore its timing speed is optically tunable by adjusting the photoconverting laser intensity. We characterized this light-driven timer method both in vitro and in vivo and applied it to image spatiotemporal distributions of several proteins with different lifetimes. This novel timer method thus offers a flexible "ruler" for studying temporal hierarchy of spatially ordered processes with exquisite spatial-temporal resolution.