Dehydrocoelenterazine as an intracellular source of coelenterazine for nuclear bioluminescence imaging with Renilla luciferase.

Coelenterazine (CTZ) is widely used as a substrate for marine bioluminescence reactions. Here, we demonstrate that dehydrocoelenterazine (dCTZ), a dehydrogenated form of CTZ, serves as an intracellular source of CTZ in mammalian cells. Fluorescence imaging revealed that dCTZ readily permeates the plasma membrane and is converted to CTZ inside cells, resulting in enhanced fluorescence signals in both the cytoplasm and the nucleus. Based on CTZ fluorescence analysis, the apparent membrane permeability of dCTZ was estimated to be more than 4.6-fold higher than that of CTZ. Furthermore, intracellular bioluminescence imaging of Renilla luciferase (RLase) and Gaussia luciferase (GLase) in mammalian cells was successfully performed using dCTZ. In RLase-expressing cells, the average maximum luminescence intensity (Imax) of CTZ converted from dCTZ (dCTZ-derived CTZ) was approximately 3.1-fold higher than that obtained with CTZ. Notably, nuclear-targeted RLase and endoplasmic reticulum-Golgi apparatus (ER-Golgi)-localized GLase were efficiently visualized in mammalian cells using dCTZ. These results indicate that dCTZ is a useful source of CTZ for CTZ-dependent intracellular bioluminescence imaging in living mammalian cells.