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Published on: September 7, 2012
Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
Thibaud T Renault1, Mark P A Luna-Vargas2, Jerry E Chipuk3
1Department of Oncological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; The Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; Department of Dermatology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA; Helmholtz Centre for Infection Research, Braunschweig, Germany; Max Planck Institute for Infection Biology, Charitéplatz 1, Campus Charité Mitte, Berlin, Germany.
Abstract:
The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is "Liver mitochondria isolation" in which the liver is dissected from mice to obtain mitochondria. "Mitochondria labeling with JC-1 and size fractionation" is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the "Real-time MOMP measurements" protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015).
Insights
Investigating mitochondrial outer membrane permeabilization (MOMP) requires isolated mitochondria. This study presents protocols for liver mitochondria isolation, labeling, size fractionation, and real-time MOMP measurements for studying apoptosis regulators.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- The mitochondrial pathway of apoptosis is crucial for cell death.
- Investigating mitochondrial outer membrane permeabilization (MOMP) is complex due to multi-factor regulation.
- Cellular models have limitations for studying MOMP regulation.
Purpose of the Study:
- To present complementary procedures for real-time investigation of MOMP regulators.
- To enable deconstruction of MOMP into individual steps using isolated systems.
- To study the role of BCL-2 family proteins and mitochondrial shape in apoptosis.
Main Methods:
- Liver mitochondria isolation from mice.
- Mitochondria labeling with JC-1 and size fractionation.
- Real-time MOMP measurements on isolated mitochondria.
Main Results:
- Established protocols for isolating and characterizing mitochondria.
- Enabled real-time monitoring of MOMP.
- Provided a system to study MOMP regulators in vitro.
Conclusions:
- Isolated mitochondria systems are effective for studying MOMP.
- The presented protocols facilitate in-depth analysis of apoptosis regulation.
- Mitochondrial membrane shape plays a role in MOMP.
