Mitophagy is a cellular process that selectively removes damaged, old or dysfunctional mitochondria. Defective mitophagy is thought to contribute to normal aging and to various neurodegenerative and cardiovascular diseases. Previous methods used to detect mitophagy in vivo were cumbersome, insensitive and difficult to quantify. We created a transgenic mouse model that expresses the pH-dependent fluorescent protein mt-Keima in order to more readily assess mitophagy. Keima is a pH-sensitive, dual-excitation ratiometric fluorescent protein that also exhibits resistance to lysosomal proteases. At the physiological pH of the mitochondria (pH 8.0), the shorter-wavelength excitation predominates. Within the acidic lysosome (pH 4.5) after mitophagy, mt-Keima undergoes a gradual shift to longer-wavelength excitation. In this protocol, we describe how to monitor mitophagic flux in living cells over an 18-h time frame, as well as how to quantify mitophagy using the mt-Keima probe. This protocol also describes how to use confocal microscopy to visualize mitophagy in living tissues obtained from mt-Keima transgenic mice. With this protocol, the mt-Keima probe can reliably be imaged within the first 60 min after tissue collection. We also describe how to apply mt-Keima with stimulated emission depletion (STED) microscopy, which can potentially provide substantially higher-resolution images. Typically, the approximate time frame for time-lapse fluorescence imaging of mt-Keima is 20 h for living cells. For confocal analysis of tissue from an mt-Keima mouse, the whole procedure generally takes no longer than 60 min, and the STED imaging usually takes <2 h.
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We are grateful to Z.-X. Yu (NHLBI Pathology Core) and P. McCoy (NHLBI FACS Core) for experimental assistance. We thank R. Youle for the Parkin-overexpressing HeLa cell line, and A. Miyawaki for the original mt-Keima construct. This work was supported by Intramural NIH funds and a Leducq Transatlantic Network grant to T.F.
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Images were obtained at 15-min intervals over an 18-h period. There is only a modest level of mitophagy observed, indicating little evidence of laser-induced photo-damage.
Confocal time-lapse of a HeLa cell line with stable expression of mt-Keima and Parkin treated with FCCP (5 μM) and oligomycin (5 μM).
Images were obtained at 15-min intervals over an 18-h period. An increase in mitophagy begins early, and this treatment eventually results in the near total loss of green mt-Keima fluorescence and a marked increase in overall red mt-Keima fluorescence.
STED time-lapse of mt-Keima mouse liver imaged ex vivo at 6,000 lines per second for 7 min with a 480-nm excitation laser.
Mapped pseudocolor was used to assess hepatocyte mitochondria morphology and dynamics. Little evidence of STED-induced photo-bleaching is observed.