title: MitoTimer: a novel tool for monitoring mitochondrial turnover.
abstract: 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.
authors.affiliation: San Diego State University; San Diego, CA USA.
authors.forename: Genaro
authors.initials: G
authors.lastname: Hernandez
authors.forename: Christine
authors.initials: C
authors.lastname: Thornton
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Stotland
authors.forename: Diana
authors.initials: D
authors.lastname: Lui
authors.forename: Jon
authors.initials: J
authors.lastname: Sin
authors.forename: Jennifer
authors.initials: J
authors.lastname: Ramil
authors.forename: Najib
authors.initials: N
authors.lastname: Magee
authors.forename: Allen
authors.initials: A
authors.lastname: Andres
authors.forename: Giovanni
authors.initials: G
authors.lastname: Quarato
authors.forename: Raquel S
authors.initials: RS
authors.lastname: Carreira
authors.forename: M Richard
authors.initials: MR
authors.lastname: Sayen
authors.forename: Roland
authors.initials: R
authors.lastname: Wolkowicz
authors.forename: Roberta A
authors.initials: RA
authors.lastname: Gottlieb
mesh_terms: D000818:Animals; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D063269:Mitochondrial Turnover; D050505:Mutant Proteins / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013997:Time Factors; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C000599934:MitoTimer protein
keywords: Fluorescent Timer protein; fluorescence imaging; mitochondria; mitochondrial turnover; mitophagy