A simple estimation of peroxisomal degradation with green fluorescent protein - an application for cell cycle analysis.
When nutrients are depleted from the environment, mammalian cells begin to degrade their own cytosol and organelles. This bulk protein degradation is mediated by autophagy. In this study, peroxisomes in living CHO-K1 cells were visualized by targeting the green fluorescent protein (GFP) tagged with a type 1 peroxisomal targeting signal. The nutrient-starved condition induced a decay of GFP fluorescence in the peroxisomes and autophagic inhibitors such as 3-methyladenine suppressed the decay of GFP fluorescence (13-60% of starvation). By double labeling the nuclear DNA and peroxisomal GFP, the autophagy specifically occurred at the G1 phase of the cell cycle and the autophagic inhibitors suppressed the G1 arrest. The vital stain technique with GFP is a very simple and useful marker to quantitatively estimate or to further study peroxisomal degradation.
Arai
K
K
Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences, Graduate School of Medicine, Kanazawa University, Ishikawa, Japan. arai@dbs.p.kanazawa-u.ac.jp
Ohkuma
S
S
Matsukawa
T
T
Kato
S
S
D000818:Animals; D016466:CHO Cells; D002453:Cell Cycle; D006224:Cricetinae; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014170:Transformation, Genetic
D008164:Luminescent Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D049452:Green Fluorescent Proteins