Chapter Thirty‐Two Use of Protease Inhibitors for Detecting Autophagy in Plants
Introduction
Autophagy is a process in which cells degrade their own components. In macroautophagy, a part of the cytoplasm including organelles is first enclosed by a double‐membrane‐bounded structure, initially called a phagophore and then an autophagosome, which subsequently fuses with a preexisting lysosome. The resulting structure is a lysosome containing a part of the cytoplasm, which is called an autolysosome. In yeast cells, autophagosomes fuse with the vacuole and release their inner‐membrane‐bounded structures into the vacuole. Thus, the resulting structure is the vacuole containing many membrane‐bounded parts of the cytoplasm (called autophagic bodies). In microautophagy, lysosomes and/or vacuoles directly incorporate bits of the cytoplasm without making autophagosomes. In both types of autophagy, parts of the cytoplasm taken up into lysosomes/vacuoles are eventually degraded by hydrolytic enzyme therein.
Protein is one of the major cytoplasmic components. Thus, the inhibition of vacuolar and/or lysosomal proteases with inhibitors blocks or slows down the degradation of parts of the cytoplasm and causes the accumulation of cytoplasmic materials in these organelles. In mammalian cells, the protease inhibitor leupeptin inhibits lysosomal cysteine proteases, cathepsins B and L, and accumulate parts of the cytoplasm in lysosomes (Kominami et al., 1983). Similarly, the serine protease inhibitor phenylmethanesulfonyl fluoride inhibits proteinase B in the yeast vacuole, which results in the accumulation of autophagic bodies in the vacuole (Takeshige et al., 1992). Yeast mutant cells lacking vacuolar protease activities exhibit the same phenotype. Thus, by treating cells with appropriate protease inhibitors and monitoring the accumulation of undegraded particles of cytoplasmic origin, we can analyze the presence and location of autophagy in the cells.
We have been investigating autophagy in plant cells using cultured tobacco (BY‐2) cells and root tips from Arabidopsis and barley. When BY‐2 cells at the logarithmic growth phase are transferred to a sucrose‐free culture medium and further cultured, cellular protein content decreases (Moriyasu and Ohsumi, 1996, Takatsuka et al., 2004). E‐64c (Tamai et al., 1986) and other protease inhibitors such as E‐64, antipain, and leupeptin added to the culture medium inhibit protein degradation and concomitantly cause the accumulation of autolysosomes (Moriyasu and Ohsumi, 1996). Such accumulation is not significant when BY‐2 cells are cultured in a medium containing sucrose, suggesting that autophagy is induced under nutrient‐starvation conditions (Inoue and Moriyasu, 2006). In contrast, basal autophagy occurs constitutively, irrespective of the presence or absence of sucrose in the culture media, in root cells from Arabidopsis and barley, although it is activated in a sucrose‐free medium (Inoue and Moriyasu, 2006, Moriyasu et al., 2003, Yano et al., 2007). Furthermore, parts of the cytoplasm accumulate in preexisting central vacuoles as well as possibly in newly formed lysosomes in root‐tip cells treated with a protease inhibitor (Inoue and Moriyasu, 2006, Moriyasu et al., 2003). In addition, 3‐methyladenine, a potent inhibitor of autophagy in mammalian cells (Gordon and Seglen, 1982, Seglen and Gordon, 1982), inhibits autophagy in BY‐2 cells (Takatsuka et al., 2004) and in Arabidopsis root‐tip cells (Inoue et al., 2006).
We think that using these protease inhibitors and 3‐methyladenine provides us with a model useful for analyzing autophagy in plant cells. This chapter describes protocols for detecting autolysosomes and large vacuoles containing many cytoplasmic inclusions in BY‐2 cells and in the root‐tip cells of Arabidopsis and barley by microscopy.
Section snippets
Culture of BY‐2 cells
BY‐2 cells are suspension‐cultured plant cells, which derive from tobacco (Nicotiana tabacum, Bright Yellow 2). They are subcultured in a Murashige and Skoog culture medium (MS medium, which consists of Murashige and Skoog salts mixture, 2 mg/l glycine, 100 mg/l myo‐inositol, 0.5 mg/l nicotinic acid, 0.5 mg/l pyridoxine‐HCl, and 0.1 mg/l thiamine‐HCl; Murashige and Skoog, 1962) containing 30 g/l sucrose and 0.2 mg/l 2,4‐dichlorophenoxyacetic acid. There are several ways of preparing stock
Detection of the Accumulation of Autolysosomes in BY‐2 Cells with Neutral Red and Quinacrine
Autolysosomes that accumulate by E‐64c treatment in BY‐2 cells can be observed by a light microscope with Nomarski (differential interference contrast) optics (Fig. 32.1). They can also be stained with quinacrine and neutral red (Fig. 32.2). Since these acidotropic reagents are basic by nature and can penetrate cells as their noncharged form, cells should be treated with these reagents in solutions with alkaline pH. Here we introduce our methods of vital staining of autolysosomes with neutral
Staining of Autolysosomes in BY‐2 Cells by the Use of Endocytosis Markers
In mammalian cells, the autophagic and endocytic pathways converge at the endosomes (Liou et al., 1997). A similar process occurs in plant cells (Herman and Lamb, 1992, Record and Griffing, 1988). Thus, autolysosomes in BY‐2 cells are located on the endocytic pathway, which can be traced using a fluorescent marker of endocytosis, FM 4–64 (Fig. 32.3).
Enzyme Cytochemistry for Acid Phosphatase by Light Microscopy
Acid phosphatase is localized in autolysosomes as well as in the central vacuole. But the enzyme in the central vacuole seems to be free from chemical fixation by aldehyde because the protein concentration of vacuolar sap is low. Thus, in this method, only autolysosomes appear to be stained.
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Place 1.0 of ml cell suspension (cells cultured in the sucrose‐free culture medium in the presence or absence of E‐64c for 1 day) in a 2.0‐ml microcentrifuge tube with a round bottom, and let the cells
Neutral Red and LysoTracker Red Staining to Detect Autolysosomes and Cytoplasmic Inclusions in the Central Vacuole in Plant Root‐Tip Cells
When the root tips of Arabidopsis and barley are incubated with E‐64d, an esterified and thus more membrane‐permeable form of E‐64c, cytoplasmic particles accumulate in large vacuoles, which appear to have preexisted before inhibitor treatment as well as in small vesicles, which appear to be formed de novo and to correspond to autolysosomes in BY‐2 cells. Since both preexisting vacuoles and newly formed lysosomes are acidic, the acidotropic dyes neutral red and LysoTracker Red are concentrated
ImmunoStaining of Lysosomes/Vacuoles in Barley Root‐Tip Cells
In barley root‐tip cells, it was reported that antibodies against alpha‐ and gamma‐TIP differentially bind to the membranes of various kinds of vacuoles (Paris et al., 1996). The membrane of the lysosomes/vacuoles that are accumulated by treatment with E‐64d can be stained with alpha‐TIP antibodies (Fig. 32.6).
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Treat root‐tip cells (step 5.i in the previous section) with 0.5% (w/v) Triton X‐100 for 5 min at room temperature.
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Keep them in a blocking buffer consisting of 0.25% (w/v) BSA, 0.25%
Electron Microscopy of Autolysosomes and Vacuoles Containing Cytoplasmic Inclusions in Plant Cells
In BY‐2 cells, autolysosomes can be detected as membrane vesicles containing electron‐dense particles by conventional electron microscopy (Fig. 32.7). Electron‐dense particles are scarcely seen in the central vacuole (Fig. 32.7). In contrast, in Arabidopsis and barley root cells, small vesicles containing electron‐dense particles are seen and the central vacuole also seems to have electron‐dense particles (Fig. 32.8). The former seems to correspond to autolysosomes in BY‐2 cells, whereas the
Enzyme Cytochemistry for Acid Phosphatase by Electron Microscopy
- 1
Fix cells (cells cultured in the sucrose‐free culture medium in the presence or absence of E‐64c for 1 day) overnight with 1% (w/v) glutaraldehyde and 1% (w/v) formaldehyde in 0.1 M sodium cacodylate‐HCl buffer, pH 7.2, at 4 °C.
- 2
Wash the cells three time with 8% (w/v) sucrose in 0.1 M sodium cacodylate‐HCl buffer, pH 7.2, and subsequently three times with 8% (w/v) sucrose in 50 mM acetate‐Na buffer, pH 5.0.
- 3
Keep the cells in 45.5 mM acetate‐Na buffer, pH 5.0, 3 mM lead nitrate, 10 mM
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