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J Biol Chem. 2014 Apr 25;289(17):12005-15. doi: 10.1074/jbc.M113.530527. Epub 2014 Mar 18.

A novel MitoTimer reporter gene for mitochondrial content, structure, stress, and damage in vivo.

Author information

1
From the Departments of Medicine.

Abstract

Mitochondrial dysfunction plays important roles in many diseases, but there is no satisfactory method to assess mitochondrial health in vivo. Here, we engineered a MitoTimer reporter gene from the existing Timer reporter gene. MitoTimer encodes a mitochondria-targeted green fluorescent protein when newly synthesized, which shifts irreversibly to red fluorescence when oxidized. Confocal microscopy confirmed targeting of the MitoTimer protein to mitochondria in cultured cells, Caenorhabditis elegans touch receptor neurons, Drosophila melanogaster heart and indirect flight muscle, and mouse skeletal muscle. A ratiometric algorithm revealed that conditions that cause mitochondrial stress led to a significant shift toward red fluorescence as well as accumulation of pure red fluorescent puncta of damaged mitochondria targeted for mitophagy. Long term voluntary exercise resulted in a significant fluorescence shift toward green, in mice and D. melanogaster, as well as significantly improved structure and increased content in mouse FDB muscle. In contrast, high-fat feeding in mice resulted in a significant shift toward red fluorescence and accumulation of pure red puncta in skeletal muscle, which were completely ameliorated by voluntary wheel running. Hence, MitoTimer allows for robust analysis of multiple parameters of mitochondrial health under both physiological and pathological conditions and will be highly useful for future research of mitochondrial health in multiple disciplines in vivo.

KEYWORDS:

Confocal Microscopy; Exercise; High-fat Diet; Mitochondria; Oxidative Stress; Reporter Gene; Skeletal Muscle

PMID:
24644293
PMCID:
PMC4002107
DOI:
10.1074/jbc.M113.530527
[Indexed for MEDLINE]
Free PMC Article

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