Renault Thibaud T, Luna-Vargas Mark P A, Chipuk Jerry E
Department 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.
Department 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.
Bio Protoc. 2016 Aug 5;6(15). doi: 10.21769/BioProtoc.1892.
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 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 ., 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 the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault ., 2015).
细胞凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用，并且还取决于线粒体的形状和大小。在过去的研究中，使用细胞模型来研究这种复杂的多因素相互作用如何调节线粒体外膜通透性（MOMP）的分子机制并不理想。已证明分离系统是一种将MOMP分解为各个步骤并研究MOMP调节因子各子集行为的范例。特别是，分离的线粒体是研究BCL-2家族蛋白的关键，BCL-2家族是一个复杂的促生存和促凋亡蛋白家族，直接控制细胞凋亡的线粒体途径（雷诺特等人，2013年）。在本方案中，我们描述了三种互补的程序，用于使用分离的线粒体实时研究MOMP调节因子的作用。第一个程序是“肝脏线粒体分离”，即从小鼠身上解剖肝脏以获得线粒体。第二个程序是“用JC-1标记线粒体并按大小分级分离”，该程序描述了一种标记、按大小分级分离并标准化线粒体亚群的方法。最后，“实时MOMP测量”方案允许在分离的线粒体上实时跟踪MOMP。上述程序用于确定线粒体膜形状在分离细胞和分离线粒体水平上的作用（雷诺特等人，2015年）。
