Monitoring mitophagy in neuronal cell cultures.
Monitoring mitophagy in neuronal cell cultures.
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DOI：
10.1007/978-1-61779-328-8_21
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Chu, Charleen T
中科院分区：
文献类型：
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作者：
Zhu, Jianhui;Dagda, Ruben K;Chu, Charleen T
适当控制线粒体周转对于在基础和应激条件下维持细胞能量学以及预防内源性氧化应激至关重要。整个细胞器的周转是通过巨自噬介导的，巨自噬是自噬体将线粒体递送到溶酶体进行水解降解的过程。虽然线粒体自噬可以作为自噬的非选择性上调的一部分而发生，但受损或不需要的线粒体的选择性降解（线粒体自噬）是发育、癌症和神经退行性疾病中快速增长的领域，特别是关于帕金森病。由于其动态的性质，和潜在的疾病过程的监管扰动，没有一个单一的技术是足以评估线粒体自噬。在这里，我们描述了几种互补的技术，包括电子显微镜，单细胞分析的LC3荧光斑点，和Western印迹，每个结合使用的通量抑制剂，以捕获新形成的自噬体，以监测神经元细胞中的线粒体自噬。
Proper control of mitochondrial turnover is critical for maintenance of cellular energetics under basal and stressed conditions, and for prevention of endogenous oxidative stress. Whole organelle turnover is mediated through macroautophagy, a process by which autophagosomes deliver mitochondria to the lysosome for hydrolytic degradation. While mitochondrial autophagy can occur as part of a nonselective upregulation of autophagy, selective degradation of damaged or unneeded mitochondria (mitophagy) is a rapidly growing area in development, cancer, and neurodegeneration, particularly with regard to Parkinson’s disease. Due to its dynamic nature, and the potential for regulatory perturbation by disease processes, no single technique is sufficient to evaluate mitophagy. Here, we describe several complementary techniques that include electron microscopy, single cell analysis of LC3 fluorescent puncta, and Western blot, each used in conjunction with a flux inhibitor to trap newly formed autophagosomes in order to monitor mitophagy in neuronal cells.