Shvets Elena, Elazar Zvulun
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Methods Enzymol. 2009;452:131-41. doi: 10.1016/S0076-6879(08)03609-4.
Autophagy is a major intracellular catabolic pathway induced in response to amino acid starvation. Recent findings implicate it in diverse physiological/pathophysiological events, such as protein and organelle turnover, development, aging, pathogen infection, cell death, and neurodegeneration. However, experimental methods to monitor this process in mammalian cells are limited because of the deficiency of autophagic markers. Recently, MAP1-LC3 (LC3), a mammalian homolog of the yeast ubiquitin-like (UBL) protein Atg8, has been shown to selectively incorporate into the autophagosomal membrane, thus serving as a unique bona fide marker of autophagosomes in mammals. Thus, the autophagic activity can be largely determined by GFP-LC3/LC3, predominantly associated with autophagosomes (when LC3 is conjugated to phosphatidylethanolamine), both biochemically and microscopically. However, current methods to quantify autophagic activity using LC3 are time consuming, labor intensive, and require expertise in accurate interpretation. In this chapter we describe the use of flow cytometry and fluorescence-activated cell sorting (FACS) as a new assay designed to quantify autophagy in cells stably expressing GFP-LC3. Flow cytometry is a well-established technique for performing quantitative fluorescence measurements, allowing quick, accurate, and simultaneous determination of many parameters in cell subpopulations. Here flow cytometry and FACS were used to quantify the turnover of GFP-LC3 (reflecting an autophagic flux) as a reliable and simple assay to measure autophagic activity in living mammalian cells.
自噬是一种主要的细胞内分解代谢途径，在氨基酸饥饿时被诱导。最近的研究结果表明它参与了多种生理/病理生理事件，如蛋白质和细胞器的更新、发育、衰老、病原体感染、细胞死亡和神经退行性变。然而，由于自噬标记物的缺乏，在哺乳动物细胞中监测这一过程的实验方法有限。最近，MAP1-LC3（LC3），一种酵母泛素样（UBL）蛋白Atg8的哺乳动物同源物，已被证明可选择性地整合到自噬体膜中，因此成为哺乳动物自噬体的一种独特的真正标记物。因此，自噬活性在很大程度上可以通过GFP-LC3/LC3来确定，GFP-LC3/LC3主要与自噬体相关（当LC3与磷脂酰乙醇胺结合时），无论是在生化方面还是在显微镜下。然而，目前使用LC3定量自噬活性的方法耗时、费力，并且需要专业知识来进行准确解读。在本章中，我们描述了使用流式细胞术和荧光激活细胞分选（FACS）作为一种新的检测方法，用于定量稳定表达GFP-LC3的细胞中的自噬。流式细胞术是一种成熟的技术，用于进行定量荧光测量，能够快速、准确且同时测定细胞亚群中的许多参数。在这里，流式细胞术和FACS被用于定量GFP-LC3的周转（反映自噬通量），作为一种可靠且简单的检测方法来测量活的哺乳动物细胞中的自噬活性。
