Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes
Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes
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DOI：
10.4161/auto.2229
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发表时间：
2006-01-01
期刊：
影响因子：
13.3
通讯作者：
Lemasters, John J.
中科院分区：
文献类型：
--
作者：
Rodriguez-Enriquez, Sara;Kim, Insil;Lemasters, John J.
营养剥夺后，线粒体成为自噬降解的目标，这一过程也称为线粒体自噬。在本研究中，我们使用 LysoTracker Red (LTR) 和 MitoTracker Green 来表征培养的大鼠肝细胞中自噬体增殖和线粒体自噬的动力学。营养剥夺加上胰高血糖素诱导的自噬增加了用荧光读板器评估的 LTR 摄取，以及用共聚焦显微镜评估的单个肝细胞中 LTR 标记的酸性细胞器的数量，两者均增加了 4 至 6 倍。与 MitoTracker Green (MTG) 共同负载的肝细胞的连续成像显示自噬诱导后平均线粒体消化时间为 7.5 分钟。在蛋白酶抑制剂存在下，消化时间增加了一倍以上，LTR标记的细胞器总数增加了约40%，但LTR标记的含有MTG荧光的酸性细胞器的比例保持恒定在75%左右。自噬抑制剂 3-甲基腺嘌呤、渥曼青霉素和 LY204002 将营养剥夺后 LTR 摄取的增加抑制高达 85%，证实 LTR 摄取的增加反映了自噬诱导。线粒体通透性转变 (MPT) 的特异性抑制剂环孢菌素 A 和 NIM811 也会降低 LTR 的摄取，而他克莫司（一种不抑制 MPT 的免疫抑制试剂）则没有效果。此外，c-Jun N 末端激酶 (JNK) 抑制剂 SCP25041 和 SP600125 分别阻止 LTR 摄取 47% 和 61%，但 ERK1、p38 和 caspase 抑制剂没有效果。结果表明，线粒体一旦被选择进行线粒体自噬，就会被迅速消化，并支持线粒体自噬涉及 MPT 以及通过 P13 激酶和可能的 JNK 进行信号传导的概念。
Mitochondria become targets for autophagic degradation after nutrient deprivation, a process also termed mitophagy. In this study, we used LysoTracker Red (LTR) and MitoTracker Green to characterize the kinetics of autophagosomal proliferation and mitophagy in cultured rat hepatocytes. Autophagy induced by nutrient deprivation plus glucagon increased LTR uptake assessed with a fluorescence plate reader and the number of LTR-labeled acidic organelles assessed with confocal microscopy in individual hepatocytes both by 4- to 6-fold. Serial imaging of hepatocytes coloaded with MitoTracker Green (MTG) revealed an average mitochondrial digestion time of 7.5 min after autophagic induction. In the presence of protease inhibitors, digestion time more than doubled, and the total number of LTR-labeled organelles increased about 40%, but the proportion of the LTR-labeled acidic organelles containing MTG fluorescence remained constant at about 75%. Autophagy inhibitors, 3-methyladenine, wortmannin and LY204002, suppressed the increase of LTR uptake after nutrient deprivation by up to 85%, confirming that increased LTR uptake reflected autophagy induction. Cyclosporin A and NIM811, specific inhibitors of the mitochondrial permeability transition (MPT), also decreased LTR uptake, whereas tacrolimus, an immunosuppressive reagent that does not inhibit the MPT was without effect. In addition, the c-Jun N-terminal kinase (JNK) inhibitors, SCP25041 and SP600125, blocked LTR uptake by 47% and 61%, respectively, but ERK1, p38 and caspase inhibitors had no effect. The results show that mitochondria once selected for mitophagy are rapidly digested and support the concept that mitochondrial autophagy involves the MPT and signaling through P13 kinase and possibly JNK.