Utilizing flow cytometry to monitor autophagy in living mammalian cells
Utilizing flow cytometry to monitor autophagy in living mammalian cells
复制标题
DOI：
10.4161/auto.5939
复制
发表时间：
2008-07-01
期刊：
影响因子：
13.3
通讯作者：
Elazar, Zvulun
中科院分区：
文献类型：
--
作者：
Shvets, Elena;Fass, Ephraim;Elazar, Zvulun
自噬是一种重要的细胞内分解代谢途径，参与多种生物学事件，包括对氨基酸饥饿、蛋白质和细胞器周转、发育、衰老、病原体感染和细胞死亡的反应。然而，由于缺乏自噬标记物，在哺乳动物细胞中监测这一过程的实验方法受到限制。最近，MAP 1-LC 3（LC 3），一种泛素样蛋白Atg 8的哺乳动物同源物，被证明选择性地掺入自噬体，从而作为哺乳动物中自噬体的唯一真实标记。然而，目前使用LC 3量化自噬活性的方法是耗时的，劳动密集型的，并且需要大量的经验来进行准确的解释。在这里，我们利用荧光激活细胞分选仪（FRCS）来定量GFP-LC 3的周转，作为测量活哺乳动物细胞中自噬活性的测定。我们发现，在诱导自噬的雷帕霉素，衣霉素或饥饿的氨基酸，荧光强度的GFP-LC 3的降低，在一个时间依赖性的方式。这种减少特别发生在野生型LC 3中，但不发生在突变型LC 3（G120 A）中，并且被自噬或溶酶体抑制剂抑制，表明这种信号对选择性自噬介导的LC 3向溶酶体中的递送是特异性的。通过利用该测定，我们测试了自噬过程的最低营养需求，并通过剥夺特定的单个氨基酸来确定其诱导。我们的结论是，这种方法可以成功地应用于不同的细胞系作为一个可靠的和简单的方法来量化活的哺乳动物细胞中的自噬活性。
Autophagy is a major intracellular catabolic pathway that takes part in diverse biological events including response to amino acid starvation, protein and organelle turnover, development, aging, pathogen infection and cell death. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, was shown to selectively incorporate into autophagosome, thus serving as a unique bona fide marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming, labor-intensive and require much experience for accurate interpretation. Here we took advantage of the Fluorescence Activated Cell Sorter (FRCS) to quantify the turnover of GFP-LC3 as an assay to measure autophagic activity in living mammalian cells. We showed that during induction of autophagy by rapamycin, tunicamycin or starvation to amino acids, fluorescence intensity of GFP-LC3 is reduced in a time-dependent manner. This decrease occurred specifically in wild type LC3, but not in mutant LC3(G120A), and was inhibited by autophagic or lysosomal inhibitors, indicating that this signal is specific to selective autophagy-mediated delivery of LC3 into lysosomes. By utilizing this assay, we tested the minimal nutrient requirement for the autophagic process and determined its induction by deprivation of specific single amino acids. We conclude that this approach can be successfully applied to different cell-lines as a reliable and simple method to quantify autophagic activity in living mammalian cells.