Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Carretera de Utrera Km. 1, Seville, Spain.
Apoptosis. 2011 Apr;16(4):404-24. doi: 10.1007/s10495-011-0577-1.
Microtubule cytoskeleton is reformed during apoptosis, forming a cortical structure beneath plasma membrane, which plays an important role in preserving cell morphology and plasma membrane integrity. However, the maintenance of the apoptotic microtubule network (AMN) during apoptosis is not understood. In the present study, we examined apoptosis induced by camptothecin (CPT), a topoisomerase I inhibitor, in human H460 and porcine LLCPK-1α cells. We demonstrate that AMN was organized in apoptotic cells with high ATP levels and hyperpolarized mitochondria and, on the contrary, was dismantled in apoptotic cells with low ATP levels and mitochondrial depolarization. AMN disorganization after mitochondrial depolarization was associated with increased plasma membrane permeability assessed by enhancing LDH release and increased intracellular calcium levels. Living cell imaging monitoring of both, microtubule dynamics and mitochondrial membrane potential, showed that AMN persists during apoptosis coinciding with cycles of mitochondrial hyperpolarization. Eventually, AMN was disorganized when mitochondria suffered a large depolarization and cell underwent secondary necrosis. AMN stabilization by taxol prevented LDH release and calcium influx even though mitochondria were depolarized, suggesting that AMN is essential for plasma membrane integrity. Furthermore, high ATP levels and mitochondria polarization collapse after oligomycin treatment in apoptotic cells suggest that ATP synthase works in "reverse" mode during apoptosis. These data provide new explanations for the role of AMN and mitochondria during apoptosis.
微管细胞骨架在细胞凋亡过程中发生重排，在质膜下形成皮质结构，对维持细胞形态和质膜完整性起重要作用。然而，细胞凋亡过程中凋亡微管网络（AMN）的维持机制仍不清楚。本研究以拓扑异构酶 I 抑制剂喜树碱（CPT）诱导的人 H460 和猪 LLCPK-1α 细胞凋亡为模型，探讨 AMN 的维持机制。结果表明，在高 ATP 水平和线粒体超极化的凋亡细胞中，AMN 能够组装；相反，在低 ATP 水平和线粒体去极化的凋亡细胞中，AMN 解体。线粒体去极化后 AMN 的解体与质膜通透性的增加有关，表现为 LDH 释放增加和细胞内钙离子水平升高。利用微管动力学和线粒体膜电位的活细胞成像监测显示，AMN 在凋亡过程中持续存在，与线粒体超极化周期相吻合。最终，当线粒体发生大的去极化时，AMN 解体，细胞发生继发性坏死。紫杉醇稳定 AMN 可防止 LDH 释放和钙离子内流，即使线粒体去极化，提示 AMN 对质膜完整性至关重要。此外，凋亡细胞中寡霉素处理后高 ATP 水平和线粒体去极化表明，ATP 合酶在细胞凋亡过程中可能以“反向”模式工作。这些数据为 AMN 和线粒体在细胞凋亡中的作用提供了新的解释。
