Wolken Gregory G, Arriaga Edgar A
Department of Chemistry, University of Minnesota , 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States.
Anal Chem. 2014 May 6;86(9):4217-26. doi: 10.1021/ac403849x. Epub 2014 Apr 18.
Mitochondrial membrane potential varies, depending on energy demand, subcellular location, and morphology and is commonly used as an indicator of mitochondrial functional status. Electrophoretic mobility is a heterogeneous surface property reflective of mitochondrial surface composition and morphology, which could be used as a basis for separation of mitochondrial subpopulations. Since these properties are heterogeneous, methods for their characterization in individual mitochondria are needed to better design and understand electrophoretic separations of subpopulations of mitochondria. Here we report on the first method for simultaneous determination of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Mitochondria were isolated from cultured cells, mouse muscle, or liver, and then polarized, labeled with JC-1 (a ratiometric fluorescent probe, which indicates changes in membrane potential), and separated with CE-LIF. Red/green fluorescence intensity ratios from individual mitochondria were used as an indicator of mitochondrial membrane potential. Reproducible distributions of individual mitochondrial membrane potential and electrophoretic mobility were observed. Analysis of polarized and depolarized regions of interest defined using red/green ratios and runs of depolarized controls allowed for the determination of membrane potential and comparison of electrophoretic mobility distributions in preparations containing depolarized mitochondria. Through comparison of these regions of interest, we observed dependence of electrophoretic mobility on membrane potential, with polarized regions of interest displaying decreased electrophoretic mobility. This method could be applied to investigate mitochondrial heterogeneity in aging or disease models where membrane potential is an important factor.
线粒体膜电位会发生变化，这取决于能量需求、亚细胞定位和形态，并且通常被用作线粒体功能状态的指标。电泳迁移率是一种反映线粒体表面组成和形态的异质性表面特性，可作为分离线粒体亚群的基础。由于这些特性是异质性的，因此需要在单个线粒体中对其进行表征的方法，以便更好地设计和理解线粒体亚群的电泳分离。在此，我们报告了第一种通过毛细管电泳结合激光诱导荧光检测（CE-LIF）同时测定单个线粒体膜电位和电泳迁移率的方法。从培养细胞、小鼠肌肉或肝脏中分离出线粒体，然后进行极化处理，用JC-1（一种比率荧光探针，可指示膜电位的变化）进行标记，并通过CE-LIF进行分离。单个线粒体的红/绿荧光强度比用作线粒体膜电位的指标。观察到了单个线粒体膜电位和电泳迁移率的可重复分布。使用红/绿比率定义的极化和去极化感兴趣区域以及去极化对照的运行分析，使得能够确定膜电位并比较含有去极化线粒体的制剂中的电泳迁移率分布。通过比较这些感兴趣区域，我们观察到电泳迁移率对膜电位的依赖性，感兴趣的极化区域显示出较低的电泳迁移率。该方法可应用于研究膜电位是重要因素的衰老或疾病模型中的线粒体异质性。
