Research-Light-Driven Fluorescent Timer For Imaging Spatial-Temporal Protein Dynamics.

Cellular function is determined by the protein dynamics both in space and time. While the protein spatial location can be imaged with great detail by fluorescent proteins, the temporal behavior of protein lifetime is much harder to measure. Harnessing the emerging photo-convertible fluorescent proteins, we have developed light-driven fluorescent timers. By illuminating cells chronically and weakly with proper light, the ratio of the fluorescence intensities between the photo-converted and the original state provides a spatial map of the protein age with sub-cellular resolution (Fig. 1). Older proteins will correspond to larger ratios. This novel fluorescent timer is driven by light instead of oxygen, monomeric, away from cellular auto-fluorescence, bright in both green and red states, photo-stable, and most importantly, optically tunable in its timing speed.


Fig. 1. Light-driven fluorescent timer for imaging spatial temporal protein dynamics in living cells.(a) Fluorescent timer can be genetically fused to any protein of interest. After expression, a weak but prolonged illumination is used to drive the timer. (b) The timer protein can change color from green to red gradually upon violet light illumination. (c) The red to green ratio increases linearly with time. The timing speed is tunable via light intensity.

We characterized this light-driven fluorescent timer both in vitro and in vivo and applied it to image spatiotemporal distributions of a variety of proteins with different lifetimes and functions including H2B, cytochrome C oxidase, paxillin, huntingtin mutant and connexin 43 (Fig. 2). This novel timer thus offers a “smart” probe for studying complex protein dynamics with exquisite spatial-temporal resolution.


Fig. 2.Light driven fluorescent timer demonstrations in mammalian HEK 293T cells.(a) The red-to-green ratio images are shown in the right column, with the color variations indicating location-dependent protein age distributions inside cells. (a) H2B-mEos2 (b) Cytochrome C oxidase-mEos2 (c) Paxillin-mEos2 (d) Huntingtin Q94-mEos2.

Relevant Papers:
1. X. Zhu, L. Zhang, Y.-T. Kao, F. Xu and W. Min. "A Tunable fluorescent timer method for imaging spatial-temporal protein dynamics using light-driven photoconvertible protein", J. Biophotonics, 2014.[PDF]