Shprung Tal, Gozes Illana
Department of Human Molecular Genetics and Biochemistry, Sackler Medical School, Tel Aviv University, Tel Aviv, 69978, Israel.
J Mol Neurosci. 2009 Mar;37(3):254-62. doi: 10.1007/s12031-008-9129-8. Epub 2008 Jul 18.
A method was developed to assess mitochondrial movement in the living cell that is dependent, in part, on microtubule and/or associating protein interactions. The leader sequence from cytochrome-c was used to drive DsRed2 fluorescent proteins to accumulate in the mitochondria, thus enabling to follow mitochondrial (cytochrome-c's) movement. For calculating the percentage of mitochondrial movement, an image-processing program was used (ImageJ). Paclitaxel, an antitumor agent, is a potent microtubule-stabilizing agent that increases the stability of tubulin polymers, inhibiting mitosis and mitochondrial activity in dividing cells. Here, we tested whether paclitaxel inhibits mitochondrial movement in pheochromocytoma cells (a neuronal model, when tested in a differentiated state). While a 2-day exposure to paclitaxel resulted in cellular toxicity (measured as inhibition of mitochondrial activity), 2-3 h exposure to paclitaxel were sufficient to inhibit mitochondrial movement as assessed in 10-20-s imaging sessions in living cells. Mitotracker deep-red staining validated the staining obtained with DsRed2-cytochrome-c and identified intact mitochondria. Results showed a significant paclitaxel dose-dependent inhibition of mitochondrial movement. This new method should enable further assessment of microtubule-interacting drugs and other cytoskeletal components for their potential influence of mitochondrial movement as a test for activity and side effects.
我们开发了一种方法来评估活细胞中的线粒体运动，该方法部分依赖于微管和/或相关蛋白的相互作用。细胞色素c的前导序列用于驱动DsRed2荧光蛋白在线粒体中积累，从而能够追踪线粒体（细胞色素c的）运动。为了计算线粒体运动的百分比，使用了一个图像处理程序（ImageJ）。紫杉醇是一种抗肿瘤药物，是一种有效的微管稳定剂，可增加微管蛋白聚合物的稳定性，抑制分裂细胞中的有丝分裂和线粒体活性。在这里，我们测试了紫杉醇是否会抑制嗜铬细胞瘤细胞（一种神经元模型，在分化状态下进行测试）中的线粒体运动。虽然暴露于紫杉醇2天会导致细胞毒性（以线粒体活性抑制来衡量），但暴露于紫杉醇2 - 3小时就足以抑制线粒体运动，这是在活细胞中进行10 - 20秒成像实验评估得出的。线粒体深染验证了用DsRed2 - 细胞色素c获得的染色结果，并识别出完整的线粒体。结果显示紫杉醇对线粒体运动有显著的剂量依赖性抑制作用。这种新方法应该能够进一步评估与微管相互作用的药物和其他细胞骨架成分对线粒体运动的潜在影响，以此作为活性和副作用的测试。
