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Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima
Published on: August 12, 2018
A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima
Nuo Sun1, Daniela Malide2, Jie Liu1
1Center for Molecular Medicine, National Heart Lung and Blood Institute, NIH, Bethesda, Maryland, USA.
Abstract:
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.
Insights
Researchers developed a new method to easily measure mitophagy, a process crucial for cell health and aging. This technique uses a fluorescent protein called mt-Keima in mice, improving how we study mitophagy in cells and tissues.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitophagy, the selective removal of damaged mitochondria, is vital for cellular health.
- Dysfunctional mitophagy is linked to aging and diseases like neurodegeneration and cardiovascular conditions.
- Existing methods for in vivo mitophagy detection are inefficient and hard to quantify.
Purpose of the Study:
- To develop a more sensitive and quantifiable method for assessing mitophagy in vivo.
- To introduce a novel transgenic mouse model expressing the pH-sensitive fluorescent protein mt-Keima.
- To establish a protocol for monitoring and quantifying mitophagic flux in living cells and tissues.
Main Methods:
- Creation of a transgenic mouse model expressing mt-Keima, a pH-dependent fluorescent protein.
- Utilizing mt-Keima's ratiometric fluorescence shift from physiological mitochondrial pH (8.0) to acidic lysosomal pH (4.5) to signal mitophagy.
- Employing time-lapse fluorescence imaging in living cells (approx. 20h) and confocal/STED microscopy for tissue analysis (within 60 min).
Main Results:
- The mt-Keima probe allows for reliable imaging of mitophagy in living cells and tissues.
- The protocol enables quantification of mitophagic flux over an 18-hour timeframe.
- High-resolution imaging of mitophagy is achievable using stimulated emission depletion (STED) microscopy.
Conclusions:
- The mt-Keima transgenic mouse model provides a robust and accessible tool for studying mitophagy.
- This method overcomes limitations of previous techniques, offering improved sensitivity and quantification.
- The developed protocol facilitates research into the role of mitophagy in aging and disease.
