Quantitative monitoring of autophagic degradation
Quantitative monitoring of autophagic degradation
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DOI：
10.1016/j.bbrc.2006.09.168
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发表时间：
2006-12-08
影响因子：
3.1
通讯作者：
Ohkuma, Shoji
中科院分区：
文献类型：
--
作者：
Kawai, Akinori;Takano, Syuichi;Ohkuma, Shoji
我们建立了一种利用线粒体基质中稳定表达绿色荧光蛋白(GFP)的CHO-K1细胞系(mtGFP-CHO)诱导自噬的定量方法。当mtGFP-CHO细胞与缺乏氨基酸和血清的培养液孵育时，随着蛋白质的降解，GFP荧光减弱。生化和形态分析有力地表明mtGFP的降解是由自噬对线粒体的批量和非选择性降解所介导的。定量测量mtGFP降解的方法是用荧光法测量GFP荧光和DNA含量，并计算DNA含量的相对GFP强度，这接近于每个细胞的平均GFP荧光。利用这种方法，我们首次证明了不同的诱导剂，如氨基酸和血清饥饿或雷帕霉素处理，以不同的动力学促进自噬。这种方法简单、相对快速，很容易适应于高通量筛选增强或抑制自噬的新药，以及调节或调节自噬的基因。(C)2006 Elsevier Inc.保留所有权利。
We developed a quantitative method for analyzing the induction of autophagy using a CHO-K1 cell line stably expressing a green fluorescent protein (GFP) in mitochondrial matrix (mtGFP-CHO). When mtGFP-CHO cells were incubated with a medium depleted of amino acids and serum, the GFP fluorescence was decreased concomitant with degradation of the protein. Biochemical and morphological analyses strongly suggested the degradation of mtGFP was mediated by bulk and non-selective degradation of mitochondria by autophagy. Quantitative measurement of the mtGFP degradation was performed by measuring the GFP fluorescence and DNA content by a fluorometric method-and calculating the relative GFP intensity of DNA content, which approximated mean GFP fluorescence per cell. Using this method, we showed for the first time that different inducers, such as amino acids and serum starvation or rapamycin treatment, promote autophagy with different kinetics. This method is easy, relatively quick, and may be easily adapted to high throughput screening for novel drugs that enhance or inhibit autophagy, and also for genes that regulate or modulate autophagy. (c) 2006 Elsevier Inc. All rights reserved.