Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Anal Chem. 2011 Mar 1;83(5):1822-9. doi: 10.1021/ac200068p. Epub 2011 Feb 10.
Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for nonbound "free" subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count, and quantify properties of individual binding events of the mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zmol of F-actin, which theoretically represents a ~2.5 μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections.
细胞骨架与线粒体之间的相互作用对于正常的细胞功能至关重要。通常，这种相互作用的评估是基于对给定样本中存在的线粒体和细胞骨架标记物的总体分析，假设这两种细胞器类型完全结合。这种测量方法存在偏差，因为它们很少考虑到未结合的“游离”亚细胞物种。在这里，我们报告了使用带有双激光诱导荧光检测（CE-LIF）的毛细管电泳来识别、分类、计数和量化线粒体和细胞骨架的单个结合事件的特性。线粒体用 DsRed2 荧光标记，而 F-肌动蛋白（一种主要的细胞骨架成分）用 Alexa488-鬼笔环肽荧光标记。在 L6 成肌细胞的典型亚细胞部分中，79%的线粒体事件没有可检测水平的 F-肌动蛋白，而其余的平均有2 zmol 的 F-肌动蛋白，这在理论上代表每个事件有2.5 µm 长的肌动蛋白丝网络。在分析之前，用胰蛋白酶处理 L6 亚细胞部分会减少可检测到 F-肌动蛋白水平的线粒体事件的比例，这是预期的线粒体表面细胞骨架蛋白的消化。单个事件的电泳迁移率分布也可用于进一步区分结合细胞骨架的线粒体事件和无细胞骨架的线粒体事件。这里描述的 CE-LIF 方法可以进一步开发，以探索细胞骨架与其他亚细胞结构的相互作用、细胞骨架不稳定药物的作用以及病毒感染的进展。
