TY  - JOUR
TI  - Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
AU  - Stankov, Metodi  
AU  - Panayotova-Dimitrova, Diana  
AU  - Leverkus, Martin  
AU  - Behrens, Georg  
VL  - 4
IS  - 7
PY  - 2014
DA  - 2014/04/05
SP  - e1089
C1  - Bio-protocol 2014;4:e1089
DO  - 10.21769/BioProtoc.1089
UR  - https://doi.org/10.21769/BioProtoc.1089
AB  - The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with the inner membrane by the lysosomal activity. Therefore GFP-LC3 and mCherry-GFP-LC3 might be visualized by conventional or confocal fluorescence microscopy (FM). In this situation mCherry-GFP-LC3 or GFP-LC3 cytoplasmic pool is visualized as a homogeneously dispersed signal and mCherry-GFP-LC3-II or GFP-LC3-II containing autophagosomes are detected as punctae formations. The number of punctae may be used as marker of autophagosomal abundance. In general we recommend counting the average number of GFP-LC3 punctae per cell.
KW  - Autophagy
KW  - MCherry-GFP-LC3
KW  - GFP-LC3
KW  - Fluorescence Microscopy
KW  - Autophagic flux
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False