Description
Mitochondrial turnover in the cell is dictated by the balance between mitophagy and mitobiogenesis. Impaired mitochondrial turnover may be reflected by cumulative damage from reactive oxygen species and is implicated in many age-related diseases. The current methods for assessing turnover provide global information on the half-life of mitochondrial proteins, which vary greatly, but do not allow for the imaging or detection of mitochondrial subpopulations undergoing biogenesis and mitophagy. We added a mitochondrial targeting sequence to the DsRed1-E5 mutant developed by Terskikh et al. and cloned it into a tetracycline-inducible construct, dubbing it "MitoTimer." This protein is incorporated into mitochondria and matures from a green fluorescent conformation to a more stable red conformation over 48 hours, allowing us to use it as a molecular clock. The objective of this thesis was to use MitoTimer to analyze mitophagy and biogenesis in isolated mitochondria and intact cells. Flow cytometry profiling of isolated mitochondria demonstrated feasibility of the approach and confirmed that fluorescence maturation was comparable to previous studies in intact cells. Using a pulse chase pulse protocol, we evaluated whether MitoTimer could be used to monitor mitophagy and mitobiogenesis in intact cells. MitoTimer expression was induced with doxycycline (pulse), followed by 48 hour washout (chase) during which time the protein matured to the red conformation before addition of treatment with drugs that stimulate mitophagy (FCCP or simvastatin) during a second pulse of doxycycline to induce a second round of MitoTimer expression (green conformation), which was incorporated into mitochondria engaged in protein import as part of biogenesis. The unexpected finding of doxycycline-independent MitoTimer expression led to the discovery that MitoTimer mRNA persisted in significant amounts for more than 48 hours and that recovery from mitophagy was accompanied by reactivation of protein translation of MitoTimer message. This is consistent with existing literature showing that nuclear-encoded mitochondrial mRNAs are present on the surface of the mitochondria and are stabilized under conditions of mitochondrial stress. Our findings additionally reveal that the nucleotide sequence corresponding to the COX8 mitochondrial targeting sequence is sufficient to achieve this post-transcriptional regulation.
