Quantitative monitoring of autophagic degradation.
We developed a quantitative method for analyzing the induction of autophagy using a CHO-K1 cell line stably expressing a green fluorescent protein (GFP) in mitochondrial matrix (mtGFP-CHO). When mtGFP-CHO cells were incubated with a medium depleted of amino acids and serum, the GFP fluorescence was decreased concomitant with degradation of the protein. Biochemical and morphological analyses strongly suggested the degradation of mtGFP was mediated by bulk and non-selective degradation of mitochondria by autophagy. Quantitative measurement of the mtGFP degradation was performed by measuring the GFP fluorescence and DNA content by a fluorometric method and calculating the relative GFP intensity of DNA content, which approximated mean GFP fluorescence per cell. Using this method, we showed for the first time that different inducers, such as amino acids and serum starvation or rapamycin treatment, promote autophagy with different kinetics. This method is easy, relatively quick, and may be easily adapted to high throughput screening for novel drugs that enhance or inhibit autophagy, and also for genes that regulate or modulate autophagy.
Kawai
Akinori
A
Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa 920-1192, Japan.
Takano
Syuichi
S
Nakamura
Nobuhiro
N
Ohkuma
Shoji
S
D000818:Animals; D001343:Autophagy / Q000502:physiology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D008856:Microscopy, Fluorescence / Q000379:methods*; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure*; D024101:Mitochondrial Proteins / Q000378:metabolism*
D024101:Mitochondrial Proteins