Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, 
dsRed, in which fluorescence shifts over time from green to red as the protein 
matures. This molecular clock gives temporal and spatial information on protein 
turnover. To visualize mitochondrial turnover, we targeted Timer to the 
mitochondrial matrix with a mitochondrial-targeting sequence (coined 
"MitoTimer") and cloned it into a tetracycline-inducible promoter construct to 
regulate its expression. Here we report characterization of this novel 
fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were 
transfected with pTRE-tight-MitoTimer and production was induced with 
doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence 
microscopy and verified by subcellular fractionation and western blot analysis. 
Dox addition for as little as 1 h was sufficient to induce MitoTimer expression 
within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The 
color-specific conformation of MitoTimer was stable after fixation with 4% 
paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent 
transition from green to red over 48 h and was amenable to analysis by 
fluorescence microscopy and flow cytometry of whole cells or isolated 
mitochondria. A second Dox administration 48 h after the initial induction 
resulted in a second round of expression of green MitoTimer. The extent of new 
protein incorporation during a second pulse was increased by administration of a 
mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and 
biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal 
new insights into mitochondrial dynamics within cells. Coupled with organelle 
flow cytometry, it offers new opportunities to investigate mitochondrial 
subpopulations by biochemical or proteomic methods.