TY - CHAP
T1 - High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy
AU - Bravo-San Pedro, J. M.
AU - Pietrocola, F.
AU - Sica, V.
AU - Izzo, V.
AU - Sauvat, A.
AU - Kepp, O.
AU - Maiuri, M. C.
AU - Kroemer, G.
AU - Galluzzi, L.
N1 - Publisher Copyright:
© 2017 Elsevier Inc.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned 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.
AB - Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned 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.
KW - Cancer
KW - High-throughput screening
KW - ImageXpress Micro XLS Widefield High-Content Analysis System
KW - Lysosomal inhibitors
KW - Nutrient deprivation
KW - Rapamycin
UR - http://www.scopus.com/inward/record.url?scp=85009895687&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2016.10.022
DO - 10.1016/bs.mie.2016.10.022
M3 - Chapter
C2 - 28253977
AN - SCOPUS:85009895687
T3 - Methods in Enzymology
SP - 71
EP - 86
BT - Methods in Enzymology
PB - Academic Press Inc.
ER -