High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy

被引:18
作者
Bravo-San Pedro, J. M. [1 ,2 ,3 ,4 ,5 ]
Pietrocola, F. [1 ,2 ,3 ,4 ,5 ]
Sica, V. [1 ,2 ,3 ,4 ,5 ,6 ]
Izzo, V. [1 ,2 ,3 ,4 ,5 ]
Sauvat, A. [2 ,3 ,4 ,5 ,7 ]
Kepp, O. [2 ,3 ,4 ,5 ,7 ]
Maiuri, M. C. [1 ,2 ,3 ,4 ,5 ]
Kroemer, G. [2 ,3 ,4 ,5 ,7 ,8 ,9 ]
Galluzzi, L. [1 ,2 ,3 ,4 ,5 ,10 ]
机构
[1] Gustave Roussy Canc Campus, Villejuif, France
[2] INSERM, U1138, Paris, France
[3] Ctr Rech Cordeliers, Equipe Labellisee Ligue Natl Canc 11, Paris, France
[4] Univ Paris Descartes Paris V, Sorbonne Paris Cite, Paris, France
[5] Univ Pierre & Marie Curie Paris VI, Paris, France
[6] Univ Paris Saclay Paris XI, Fac Med, Le Kremlin Bicetre, France
[7] Gustave Roussy Canc Campus, Metabol & Cell Biol Platforms, Villejuif, France
[8] Hop Europeen Georges Pompidou, Pole Biol, AP HP, Paris, France
[9] Karolinska Univ Hosp, Stockholm, Sweden
[10] Weill Cornell Med Coll, New York, NY 10065 USA
来源
MOLECULAR CHARACTERIZATION OF AUTOPHAGIC RESPONSES, PT A | 2017年 / 587卷
关键词
CELL-DEATH; AUTOPHAGY; MECHANISMS; INFLAMMATION; DISEASE; LC3;
D O I
10.1016/bs.mie.2016.10.022
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Macroautophagy is a specific variant of autophagy that involves a dedicated doublemembraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3(+) dots by automated fluorescence microscopy.
引用
收藏
页码:71 / 86
页数:16
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