NIH Research Festival
Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitochondrial turnover and mitophagy in vivo. To this end we recently reported (Sun et al, Molecular Cell, 60, 2015) a transgenic mouse model in which we expressed a mitochondrial targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. This reporter mouse containing the fluorescent reporter mitochondrial targeted mt-Keima allows for the in vivo assessment of mitophagy in various tissues under normal conditions using confocal microscopy. Furthermore on another scale of resolution we demonstrated that mt-Keima is suitable for imaging via super-resolution STED microscopy allowing observation of biological phenomena never before seen. Fluorescence nanoscopy, or super-resolution microscopy has become an important tool for cell biologists with several methods achieving sub-100-nm resolution by taking advantage of reversible photo-physical switching properties of fluorescent markers. Thus using the mt-Keima mouse, we can assess at nanoscale resolution in living tissues including skeletal muscle, heart, liver, adipose tissue, and kidney, how tissues mitophagy is altered following changes in diet, oxygen availability, genetic perturbations, or aging. Dual and multi-color experiments showed 3D characterization of intracellular compartments: lysosomes, endoplasmic reticulum, nucleus, plasma membrane, lipid droplets. Moreover, we also achieved STED imaging using resonant-scanning fast-time acquisition. In conclusion we demonstrated that mt-Keima mouse is a versatile tool for investigating mitophagy via fluorescence nanoscopy at 50-nm resolution.
Scientific Focus Area: Cell Biology
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