Use of protease inhibitors for detecting autophagy in plants.
In cultured tobacco (BY-2) cells, autophagy seems to be induced under nutrient-starvation conditions, whereas in root cells from Arabidopsis and barley, it occurs constitutively though is activated under nutrient starvation conditions. In both cases, protease inhibitors such as E-64, E-64c, antipain, and leupeptin block autophagy at the step of degradation of the cytoplasm enclosed in lysosomes/vacuoles, and cause the accumulation of autolysosomes (lysosomes containing parts of the cytoplasm) and/or of many cytoplasmic inclusions in the central vacuoles. Both types of autophagy are inhibited by 3-methyladenine, which is known as a potent inhibitor of autophagy in mammalian cells. Thus, using protease inhibitors and 3-methyladenine provides us with a method useful for analyzing autophagy in plant cells. This chapter describes protocols for detecting autophagic compartments in BY-2 cells and in the root-tip cells of Arabidopsis and barley by microscopy.
Moriyasu
Yuji
Y
Department of Regulatory Biology, Faculty of Science, Saitama University, Saitama, Japan.
Inoue
Yuko
Y
D000135:Acid Phosphatase / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D004705:Endocytosis / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007202:Indicators and Reagents / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron / Q000379:methods; D009499:Neutral Red / Q000378:metabolism; D059828:Plant Cells; D018517:Plant Roots / Q000166:cytology / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism*; D011796:Quinacrine / Q000378:metabolism; D036226:Seedlings / Q000166:cytology / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism
D015415:Biomarkers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011480:Protease Inhibitors; D009499:Neutral Red; D013395:Sucrose; D000135:Acid Phosphatase; D011796:Quinacrine