Measuring autophagy in stressed cells.
Measuring autophagy in stressed cells.
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DOI：
10.1007/978-1-4939-2522-3_10
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Macleod, Kay F
中科院分区：
文献类型：
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作者：
Sharifi, Marina N;Mowers, Erin E;Drake, Lauren E;Macleod, Kay F
巨噬是细胞中的一个主要分解代谢过程，用于降解蛋白质聚集体、功能失调的细胞器和细胞内的病原体，否则这些病原体就会变得有毒。自噬还通过回收降解的自噬货物为细胞产生能量和代谢物，这对细胞在应激状态下的生存特别重要。自噬通量速率的变化对细胞功能和疾病的重要性日益被认识到，在不同的实验系统中测量自噬的兴趣也相应地增长。在这里，我们描述了在该领域中用于测量自噬通量的关键方法，包括通过蛋白质印迹、荧光细胞染色和流式细胞术监测LC3的加工，以及其他自噬标志物的水平变化或翻译后修饰，如p62/Sqstm1和ATG5-Atg12结合物。我们还描述了哪些细胞压力可以用来诱导自噬，以及如何控制自噬通量的变化，而不是抑制通量。最后，我们详细介绍了可用于监测体内自噬的技术。
Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5–Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.