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Chapter Thirty‐One Chimeric Fluorescent Fusion Proteins to Monitor Autophagy in Plants
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Chimeric Fluorescent Fusion Proteins to Monitor Autophagy in Plants Ken Matsuoka* Contents 1. Introduction 2. Fluorescent Proteins and Autofluorescence in Plant Cells and Organelles 3. Visual Detection of Autophagosomes and Autophagic Bodies Using Fluorescent Protein-Tagged ATG8 3.1. General note for the visual detection of autophagosomes in tobacco BY-2 cells expressing YFP-NtAtg8a under nutrient starvation conditions 3.2. Preparation of culture medium and standard culture conditions 3.3. Preparation of sugar-starved medium 3.4. Preparation of phosphate or nitrogen-starved medium 3.5. Nutrient starvation and detection of autophagosomes in living cells 3.6. Colocalization analysis of autophagosomes and other cellular structures 4. Visual Detection of Autophagic Degradation Using Fluorescent Protein-Tagged Synthetic Cargo 4.1. Aggregated fluorescent protein as a reporter protein 4.2. Expression and detection of autophagic degradation in tobacco BY-2 cells 4.3. Expression and detection of autophagic degradation in leaves of Arabidopsis plants expressing the cytochrome b5-DsRed fusion protein 5. Quantification of Fluorescent Fusion Proteins After Separation by Gel Electrophoresis
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Faculty of Agriculture, Kyushu University, Fukuoka, Japan
Methods in Enzymology, Volume 451 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)03231-X
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2008 Elsevier Inc. All rights reserved.
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5.1. Protein sample preparation, separation by SDS-PAGE, and detection of the DsRed fusion protein from transformed tobacco BY-2 cells Acknowledgments References
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Abstract Autophagy is induced under nutrient-deficient conditions in both growing tobacco BY-2 cultured cells as well as Arabidopsis and others intact plants. The fluorescent protein-tagged structural protein for autophagosomes, the Atg8 protein, allows nondestructive detection of autophagy induction in plant cells and tissues by fluorescence microscopy. Using this technique, the general operation of autophagy in growing root cells has been observed. A synthetic cargo protein for autophagy consisting of cytochrome b5 and the red fluorescence protein, DsRed, allows for the quantitative assay of autophagy in tobacco cells. This chapter describes methods for detecting autophagy in these plant cells using fluorescent protein fusions in situ with light microscopy, as well as quantification of autophagy.
1. Introduction Real-time detection of autophagic events is important for the molecular dissection of the process of autophagy. Fluorescent proteins, such as green fluorescent protein (GFP), red fluorescent protein (RFP), and derivatives of these proteins are now widely used to detect intracellular transport events. Yet special caution is necessary to use such fluorescent proteins in differentiated plant cells, including photosynthetic cells in green leaves and cells accumulating secondary metabolites; these cells have compounds that are highly fluorescent and tend to interfere with the detection of fluorescent proteins. The detection of autophagy induction can be done by monitoring the presence of autophagosomes in cells. The yeast Atg8 protein and its orthologs in higher organisms are widely used to detect autophagosomes, after tagging this protein with GFP and its derivatives (see, e.g., Mizushima and Kuma, 2008). Using this technique in combination with other methods, crucial roles for plant autophagy have been uncovered. These include degradation of oxidized proteins during oxidative stress (Sla´vikov et al., 2005; Xiong et al., 2007), disposal of protein aggregates (Toyooka et al., 2006), and possibly removal of damaged proteins and organelles during normal growth conditions as a housekeeping function (Sla´vikov et al., 2005; Yano et al., 2007). Quantification of autophagic degradation is also important for the elucidation of the autophagic process and its regulation. In yeast cells, activation of an alkaline phosphatase precursor, Pho8D60, dependent on the activity of
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vacuolar proteases was used to quantify autophagy and to isolate mutants in this pathway (Noda et al, 1995; see the chapter by Noda and Klionsky in this volume). In plants, we have developed a system to use fluorescent artificial cargo proteins to quantify autophagy in plant cells (Toyooka et al., 2006). In this chapter, I summarize the recent use of fluorescent proteins for the analysis of autophagy in plant cells and provide protocols and notes on these methods.
2. Fluorescent Proteins and Autofluorescence in Plant Cells and Organelles Developed plant cells tend to have a large quantity of fluorescence compounds, such as chlorophyll and lignin, which tend to interfere with the detection of fluorescent proteins. Chlorophyll is an essential pigment for photosynthesis that produces the green color in leaves. Although this compound is only found in chloroplasts, the volume of this organelle in photosynthetic cells is quite large, and accordingly this affects the analysis of fluorescent proteins in plant photosynthetic cells. In addition, chloroplasts in living cells change intracellular location and angles when cells are illuminated with visible light (Wada M et al., 2003). For example, when photosynthetic cells are illuminated with white light when adjusting the focus of the microscope, the chloroplasts tend to alter their orientation so that they face the plane of the light source to absorb the maximum intensity of light. Such behavior of chloroplast as well as the nature of chlorophyll, which emits red fluorescence with broad excitation wavelengths, tends to prevent the detection of intracellular structures when using a fluorescence microscope. The bottom wavelength of the excitation spectrum is approximately 480 nm (Fig. 31.1). This wavelength is similar to the excitation peak wavelength of GFP and yellow fluorescent protein (YFP). Thus, these proteins can be used without significant loss of detection sensitivity (see Fig. 31.1). Likewise, the red fluorescent protein DsRed has three-excitation peak wavelengths and one of the shortest is approximately 480 nm (Campbell et al., 2002). Thus, this protein can also be used with appropriate filter sets without interfering with the fluorescence of chlorophylls. The more problematic cells and tissues are lignified tissues and cells accumulating phenolic compounds. Lignin and related compounds have a high intensity of fluorescence with a broad excitation and emission spectrum (Willemse, 1989). Induction of a stress response induces formation of lignin and related compounds in plants. For example, application of the stress-related phytohormone methyl-jasmonate to tobacco BY-2 cells, which usually display very little fluorescence, induces the synthesis of phenolic compounds and the accumulation of a large quantity of fluorescent
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Relative intensity
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Figure 31.1 Relative intensity of excitation spectra of rice leaf at 700-nm emisson (gray line), EGFP (thick line) and DsRed (thin line).
pigments in vacuoles (Galis et al., 2006; Matsuoka and Galis, 2006). In this case, fluorescent proteins cannot be used to monitor cellular events because of the intense fluorescence of the central vacuoles, which comprise more than 90% (w/v) of the cell volume. Therefore, caution with regard to autofluorescence is needed to monitor autophagy in plant cells, as vacuoles are the lytic organelle for autophagic degradation in plants (Bassham 2007).
3. Visual Detection of Autophagosomes and Autophagic Bodies Using Fluorescent Protein-Tagged ATG8 Plants generally have multiple Atg8 proteins. Arabidopsis have nine Atg8 genes that are expressed differentially in different organs (Slavikova et al., 2005; Yoshimoto et al., 2002), and tobacco BY-2 cells express at least five distinct Atg8 orthologs (Toyooka and Matsuoka, 2006). Cellular functions and biochemical evens of these proteins as well as many of the homologs of other Atg proteins in plants during autophagy progression are similar to those observed in yeast and mammals (Doelling et al., 2002; Fujioka et al., 2008; Ketelaar et al., 2004; Phillips et al., 2008; Su et al., 2006; Thompson et al., 2005; Xiong et al., 2005). Thus, these proteins function as structural proteins for the autophagosome generation and can be used to detect autophagosomes and autophagic transport to the vacuoles in tobacco BY-2 cells, Arabidopsis protoplasts or Arabidopsis root cells after tagging GFP or YFP at their N terminus (Contento et al., 2005; Toyooka et al., 2006; Toyooka and Matsuoka, 2006; Yano et al., 2007; Yoshimoto et al., 2004). Essentially no difference in autophagosome detection was observed using different types of fluorescent protein tags in both Arabidopsis root cells and tobacco BY-2 cells under starvation conditions (Toyooka and Matsuoka, 2006;
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Yoshimoto et al., 2004). Thus, chimeric proteins consisting of one of the Atg8 family members and GFP or YFP expressed under the control of a constitutive promoter can be used to monitor autophagy by detecting the fluorescence of such proteins. The following are the protocols for monitoring autophagy induction in transformed tobacco cells that are easy to use for the detection of autophagosomes within a day of nutrient starvation.
3.1. General note for the visual detection of autophagosomes in tobacco BY-2 cells expressing YFP-NtAtg8a under nutrient starvation conditions Stably transformed tobacco BY-2 cells are subcultured weekly in 95 ml of culture medium as described previously (Toyooka et al., 2006). As cells reach the stationary phase of growth within a week due to the limitation of phosphate and nitrogen sources in the medium, autophagy is already induced in cells at such a growth phase (Toyooka et al., 2006). Thus, cells in logarithmic growth phase (i.e., 3 days after subculture) should be used in all experiments.
3.2. Preparation of culture medium and standard culture conditions 1. Dissolve 30 g of sucrose and the premixed powder Murashige & Skoog salts (Wako Pure Chemicals, Osaka, Japan) in approximately 900 ml of water. 2. Add 10 ml of 20 g/l KH2PO4 (pH not adjusted). 3. Add 1 ml of 1000x vitamins (1000x ¼ 100 g/l myo-inositol, 1 g/l thiamin-HCl). 4. Add 20 ml of 2,4-D stock (10 mg/ml 2,4-dichlorophenoxyacetic acid in ethanol). 5. Adjust the pH of the solution to 5.8 with 1 M KOH, and bring the volume up to 1000 ml. 6. Pour 95 ml into a 300-ml conical flask, and cover the opening with two layers of aluminum foil. 7. Autoclave at 121 C for 15 min, cool down to room temperature, and store in the dark. 8. Once a week, transfer 1.5 ml of culture at the stationary phase to 95 ml of fresh medium, and culture at 26.5 C 1 C with rotation of 130 rpm. In some case transformed cells grow slower than nontransformed cells. In this case, a larger volume of culture (up to 5 ml) is transferred to fresh medium.
3.3. Preparation of sugar-starved medium Use 15 g of mannitol instead of 30g of sucrose. The other conditions are the same as for the standard media described previously.
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3.4. Preparation of phosphate or nitrogen-starved medium
1. 1000x B, Mo solution: 6 g/l H3BO4, 250 mg/l Na2MoO42H2O. 2. 100x Fe, Mn, Zn solution: 1.7 g/l MnSO47H2O, 1.05 g/l ZnSO4 7H2O, 5.125 g/l FeSO47H2O, 3.75 g/l Na2EDTA. 3. 1000x trace elements: 830 mg/l KI, 25 mg/l CoCl26H2O, 25 mg/l CuSO45H2O. 4. 20x phosphate-free major salts: 33 g/l NH4NO3, 38 g/l KNO3, 8.8 g/l CaCl22H2O, 7.4 g/l MgSO47H2O. 5. 20x nitrogen-free major salts: 3.4 g/l KH2PO4, 8.8 g/l CaCl22H2O, 7.4 g/l MgSO47H2O. 6. Mix approximately 900 ml of water, 50 ml of appropriate major salts (either phosphate- or nitrogen-free), 1 ml of B, Mo solution, 10 ml of Fe, Mn, Zn solution, 1 ml of trace elements, 30 g of sucrose, 1ml of 1000x vitamins, and 20 ml of 2,4-D stock, and adjust to pH 5.8 with KOH. Sterilize as previously.
3.5. Nutrient starvation and detection of autophagosomes in living cells Transformed BY-2 cells expressing YFP-Atg8 at the logarithmic growth phase are transferred into nutrient-free MS medium to induce autophagy. 1. Place 10–50 ml of a 3-day-old culture into a 50-ml conical tube. 2. Centrifuge at 100 g for 5 min to pellet cells. 3. Remove the supernatant fraction and add the original volume of medium lacking one of the nutrients. Suspend the cells with gentle shaking. 4. Pellet the cells again by centrifugation to wash out the original medium. 5. Remove the supernatant fraction, and then add the same volume of medium lacking one of the nutrients. Suspend the cells. 6. Transfer the cell suspension into a conical flask of appropriate volume. We use 100-ml flasks for 10–30 ml of culture, 200-ml flasks for 30–60 ml of culture, and 300-ml flasks for 60–95 ml of culture. 7. Shake flasks using a rotary shaker at 26.5 C at a rotation speed 130 rpm. 8. After 24 h, an aliquot of the cell suspension is placed on a slide glass, covered with cover glass and monitored for the presence of autophagosomes using an epifluorescence microscope or laser-scanning confocal microscope with GFP or FITC filter sets. Dotted structures of 100– 1000 nm in diameter, which are moving slowly in the cells, are the autophagosomes (Fig. 31.2). Note: The expression level of YFP-NtAtg8a in tobacco BY-2 cells is not very high. Therefore, when observing the cells with an epifluorescence microscope equipped with the usual mercury lamp, a high-sensitivity CCD camera is necessary to detect autophagosomes. We routinely use an
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Figure 31.2 Autophagosome formation in transformed tobacco BY-2 cells expressing YFP-NtAtg8a. Logarithmic phase tobacco BY-2 cells expressing theYFP-NtAtg8a construct under the control of the CaMV35S promoter (left) and 24 h after induction of autophagy (right). Images were collected using an Olympus IX70 inverted fluorescence microscope with NBA filter sets with a Roper CoolSNAP HQ camera.The dotted structures in the left images are the autophagosomes.
Olympus IX70 or IX81 fluorescence microscope equipped with a Roper CoolSNAP HQ camera. Autophagy inhibitors 3-methyladenine or E-64 can be included during the incubation for a negative control. For details, see Takatsuka et al. (2004) or the chapter by Moriyasu and Inoue in this volume.
3.6. Colocalization analysis of autophagosomes and other cellular structures Colocalizaton studies of autophagosomes and other cellular structures are important for the analysis of autophagy targets under various induction conditions. However, as autophagosomes are transient structures during the entire autophagic degradation process, it is difficult to quantify the association of autophagosomes and cellular structures in living cells. Therefore we use acetone-fixed cells retaining YFP fluorescence (or YFP fluorescence and other fluorescent proteins in cells expressing two different fluorescent fusion proteins) to analyze the colocalization. 1. Cool 100% acetone to 20 C in a freezer. 2. Transfer 2 ml of autophagy-induced BY-2 cell culture into a 15-ml conical tube. 3. Centrifuge at 1000 g for 5 min. Remove culture medium and discard. 4. Suspend cells in 5 ml of PBS (10 mM sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.5). 5. Centrifuge as previously and remove as much PBS as possible. 6. Add 5 ml of cold acetone, mix, and keep in a freezer for 10 min. During this incubation most of the cells sediment to the bottom. 7. Remove acetone using a Pasteur pipette. 8. Wash cells by adding 10 ml of PBS, suspend, and collect the cells by centrifugation.
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9. Repeat PBS wash twice. 10. Mount cells on slides and analyze the colocalization of the YFP signal and DsRed or other fluorescent protein signals using a fluorescence microscope. Alternatively, fixed cells are further stained with antibodies as described previously to localize organelles (Toyooka et al., 2006).
4. Visual Detection of Autophagic Degradation Using Fluorescent Protein-Tagged Synthetic Cargo Detection of autophagosomes using fluorescent protein-tagged Atg8 is an easy and reproducible method for the detection of autophagy. Fluorescent autophagosomes tagged with Atg8 fusion proteins are seen as dots. Yet Atg8 and its orthologs tend to also form intracellular dot structures that are not related to autophagy (Kuma et al., 2007). Therefore, an alternative method is needed to confirm that autophagy is actually taking place. One of the systems to detect autophagic degradation is to monitor the relocation of cargo protein from the cytoplasm to the vacuole. As plant vacuoles occupy most of the cell volume, relocation of cytoplasmic protein to the vacuoles can be easily monitored using light microscopy. This strategy was used to detect transport of GFP-Atg8 from the cytoplasm to the vacuole in Arabidopsis root cells (Yoshimoto et al., 2004), as well as to monitor transport of a synthetic autophagic cargo from the cytoplasm to the vacuole in tobacco BY-2 cells (Toyooka et al., 2006). In this section, detailed information for the use of synthetic fluorescent cargo proteins to monitor autophagic degradation is discussed.
4.1. Aggregated fluorescent protein as a reporter protein Protein aggregates can be easily formed when a protein contains two or more oligomerization domains. Overexpression of such proteins tends to cause aggregates to form in the cell. One such protein is a fusion protein of cytochrome b5 and the original DsRed protein (Toyooka et al., 2006). Cytochrome b5 is an integral endoplasmic reticulum membrane protein, which tend to form into an octamer after solublization (Calabro et al., 1976). DsRed is a tetrameric red fluorescent protein (Baird et al., 2000). Thus, the expression of a fusion construct of these two proteins causes the self-association of a protein with two different affinity sites, namely from both cytochrome b5 and DsRed, and generates protein aggregates that emit strong red fluorescence after excitation with green light in tobacco BY-2 cells (Toyooka et al., 2006). The tetrameric nature of DsRed is essential for the generation of the aggregates, as a fusion protein of cytochrome b5
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and a monomeric mutant of DsRed causes the exclusive targeting of this fusion protein to the endoplasmic reticulum (supplemental figure in Toyooka et al., 2006).
4.2. Expression and detection of autophagic degradation in tobacco BY-2 cells 1. Generate a cytochrome b5-DsRed fusion protein construct (Toyooka et al., 2006) under the control of a strong promoter, such as the tandem 35S promoter (Matsuoka and Nakamura, 1991) and introduce into tobacco BY-2 cells. 2. Culture stably transformed cells as described above (Preparation of culture medium and standard culture conditions) and observe red fluorescence using a fluorescence microscope with a RFP filter set. 3. At log phase, most of the cells contain punctate protein aggregates with red fluorescence (Fig. 31.3). 4. Induce autophagy as previously using sugar-, phosphate- or nitrogenfree media. Red fluorescence can be seen emitting from most parts of the cells, indicating that some of the RFP fusion protein is targeted to the vacuole and converted to a soluble form (see Fig. 31.3). 5. If required, vacuolar membrane can be stained with a styryl dye, FM 1-43 (Invitrogen, T35356), which gives green fluorescence under blue light excitation (Emans et al., 2002). In this case, an aliquot of stock solution of FM 1-43 in DMSO (20 mM) was mixed with the autophagy-induced culture to a final concentration of 20 mM, and further incubated for 2 h at 26.5 C with shaking.
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Figure 31.3 Expression and aggregate formation of cytochrome b5-DsRed fusion protein in tobacco BY-2 cells and vacuolar targeting of the cytochrome b5-DsRed fusion protein into vacuoles. Logarithmic phase tobacco BY-2 cells expressing a cytochrome b5-DsRed construct under an enhancer duplicated derivative of the CaMV35S promoter (left) and 24 h after the induction of autophagy (right). Images were collected using an Olympus IX70 inverted fluorescence microscope withWIG filter sets equipped with an Olympus DP70 digital camera.
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6. Likewise, the lumen of the vacuoles can be stained with BCECF to emit green fluorescence (Matsuoka et al., 1997). In this case, an aliquot of stock solution of BCECF-AM in DMSO (Invitrogen, B3051) was mixed with the autophagy-induced culture to a final concentration of 6 mM, incubated for 5 min at room temperature and washed the cells in the culture with flesh medium. Fluorescence of BCECF was monitored using fluorescence microscopes with a standard GFP filter setting. Note: Roots of transformed Arabidopsis plants can be analyzed with the same setting of the microscope.
4.3. Expression and detection of autophagic degradation in leaves of Arabidopsis plants expressing the cytochrome b5-DsRed fusion protein Green leaves contain a lot of chlorophyll and other pigments for photosynthesis. Thus, choosing the proper wavelengths of both excitation and fluorescence emission is necessary to obtain clear images of RFP fusion proteins. 1. Transform Arabidopsis plants using a dipping method (Zhang et al., 2006) and collect seeds from kanamycin-resistant T1 plants. 2. Germinate kanamycin-resistant seeds on Murashige-Skoog plate as described (Zhang et al., 2006). 3. Observe fluorescence of DsRed in leaves using a Zeiss LSM 510 META or other confocal laser scanning microscope with variable absorption wavelength. Choose 488-nm laser excitation and 580-nm fluorescence emission. Chloroplasts show weak fluorescence at 580 nm under blue light excitation (Terao et al., 1995). Thus, this condition allows the detection of fluorescent aggregates in the presence of weak background fluorescence from chloroplasts in the leaves of transformants, whereas no such structures were seen in control plants without expressing cytochrome b5-DsRed fusion protein (Fig. 31.4). 4. Detach leaves from seedlings and incubate with water for 24 h in the dark to induce autophagy. Under this condition a decrease in the red fluorescent puncta is observed. For control, leaves are incubated with Murashige-Skoog medium containing 1% (w/v) sucrose. Note: Recording of DsRed fluorescence in roots can also be done with the same condition as for leaves. The emission fingerprinting protocol of the Zeiss LSM 510 META microscope allows more clear separation of chloroplasts and aggregates. However, patterns of chloroplast fluorescence change under growth and starvation conditions. Thus, as a control, the fluorescence patterns of chloroplasts should be determined experimentally using nontransformed Arabidopsis leaves grown or incubated exactly under the same condition as for leaves expressing the cytochrome b5-DsRed fusion protein.
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Figure 31.4 Expression and aggregate formation of a cytochrome b5-DsRed fusion protein in Arabidopsis plants. Upper panels show transgenic and control plants. Middle panels show red fluorescence emitted from Arabidopsis leaves after excitation with a 488-nm laser and 580-nm florescence emission using a Zeiss LSM 510 META microscope. Punctate signals of the aggregates of the cytochrome b5-DsRed fusion protein are apparent in the transformants expressing the cytochrome b5-DsRed fusion protein. Lower panels show red fluorescence emitted from Arabidopsis roots. Arrowheads in middle and lower panels indicate some of the aggregates of the cytochrome b5-DsRed fusion protein.
5. Quantification of Fluorescent Fusion Proteins After Separation by Gel Electrophoresis It is not sufficient to monitor autophagy by fluorescence microscopy; it is also necessary to quantify autophagic activity for the full molecular dissection of autophagy. Here, I describe a method to quantify autophagic degradation using the chimeric fusion proteins described previously. A basic background on the quantification of autophagy is that, when proteins are transported to a lytic environment, such as the lumen of the vacuole, these proteins are degraded partially or completely by proteases in the organelle. The relatively stable nature of the fluorochromes of DsRed and GFP in the presence of SDS allows us to detect the fusion proteins or derivatives after separation by SDS-polyacrylamide gel electrophoresis (Baird et al., 2000; Shimizu et al., 2005). Thus, vacuolar delivery of
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cytochrome b5-DsRed and conversion of this protein to a soluble form through proteolytic processing by vacuolar proteases can be detected as the change of relative molecular mass of the RFP in transformed plant cells expressing the fusion construct.
5.1. Protein sample preparation, separation by SDS-PAGE, and detection of the DsRed fusion protein from transformed tobacco BY-2 cells 1. Culture stably transformed tobacco BY-2 cells expressing the fusion protein as previously and induce autophagy. 2. Transfer 1 ml of cells into a 15-ml round-bottomed polypropylene tube (e.g., Falcon #352063). 3. Spin the tube at 1000 g for 5 min. 4. Collect and measure the volume of the supernatant fraction. 5. Add 1 ml of PBS (pH 7.5 at room temperature) to the cell pellet. Suspend by gentle shaking. 6. Centrifuge as previously, remove supernatant fraction, and discard. 7. Add 1 ml of PBS to cell pellet and suspend cells. Chill on ice for at least 5 min (up to 1 h without any effect on the processing). 8. Sonicate the cell suspension using a probe-type sonicator (UR-20P, TOMY Seiko, Tokyo, Japan) for 30 s, twice with a 1-min interval between sonications. 9. Transfer the disrupted cell suspension into a 1.5-ml microcentrifuge tube and centrifuge at 1000 g for 5 min at 4 C. 10. Collect the supernatant fraction. The supernatant fraction may be stored at 20 C at this point if required. 11. Mix the supernatant fraction (disrupted cells) and an equal volume of 2x SDS-PAGE loading buffer [0.01% (w/v) bromophenol blue, 4% (w/v) SDS, 20% (w/v) glycerol, 100 mM Tris-HCl, pH 8.8]. 12. Incubate at room temperature for 10–30 min and apply to a 12.5% (w/v) polyacrylamide minigel of 0.75 mm thickness using the Hoefer SE 260 Mighty Small system or equivalent. 13. Apply 25 mA current per gel until the dye front reaches the bottom of the gel. 14. After the separation of proteins, the gel is separated from the plates and placed into a plastic bag. 15. Put the plastic bag onto the glass stage of a Typhoon 8600 or 9400 image scanner (GE Health Care, London). 16. Scan the gel image using a 532-nm excitation laser at 650V through a 580BP30 emission filter, which allows the detection of DsRed. A typical image of the detection of autophagic degradation of the cytochrome b5-DsRed fusion protein is shown in Fig. 31.5.
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Figure 31.5 Processing of cytochrome b5-DsRed fusion protein under starvation conditions. Rapidly growing transformed tobacco BY-2 cells expressing the cytochrome b5-DsRed fusion protein were incubated for 24 or 48 h in complete culture medium or medium lacking either sucrose, nitrogen containing compounds or phosphate. Proteins prepared from cells were separated by SDS-PAGE, and DsRed-related proteins were detected using a Typhoon 8600 image analyzer. Open arrowhead indicates intact cytochrome b5-DsRed protein. Closed arrowhead indicates processed cytochrome b5-DsRed protein accumulated in the vacuoles.
Note: Prolonged incubation of protein samples with sample buffer or heating samples abolishes the fluorescence. The same quantification procedure can be applied for transformed Arabidopsis leaves and roots. In this case tissues are homogenized with the same buffer using a motor and pestle. After scanning, gels can either be stained with an appropriate dye to detect proteins or can be used for immunoblotting. Fluorescence of GFP or YFP fusion proteins can also be detected using the same scanners with different settings (Shimizu et al., 2005). As GFP or YFP fluorochromes are unstable in acidic conditions, fusions with these proteins and Atg8 tend to lose fluorescence after targeting to vacuoles.
ACKNOWLEDGMENTS I thank Drs. Kiminori Toyooka in the RIKEN Plant Science Center and Ivan Galis in the Max Planck Institute for Chemical Biology for sharing data presented in this work. This work was supported in part by a grant-in-aid for scientific research from MEXT Japan.
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Terao, T., Yamashita, A., and Satake, K. (1985). Chlorophyll b-deficient mutants of rice : I. Absorption and fluorescence spectra and chlorophyll a/b ratios. Plant Cell Physiol. 26, 1361–1367. Thompson, A., Doelling, J., Suttangkakul, A., and Vierstra, R. (2005). Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. Plant Physiol. 138, 2097–2110. Toyooka, K., Moriyasu, Y., Goto, Y., Takeuchi, M., Fukuda, H., and Matsuoka, K. (2006). Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells. Autophagy 2, 96–106. Toyooka, K., and Matsuoka, K. (2006). Autophagy and non-classical vacuolar targeting in tobacco BY-2 cells. In ‘‘Tobacco BY-2 cells: From cellular dynamics to omics’’ (T. Nagata, K. Matsuoka, and D. Inze, eds.) Vol. 58, pp. 167–180. Springer Verlag, Berlin. Wada, M., Kagawa, T., and Sato, Y. (2003). Chloroplast movement. Annu. Rev. Plant Biol. 54, 455–468. Willemse, M. T. M. (1989). Cell wall autofluorescence. In ‘‘Physico-chemical characterisation of plant residues for industrial and feed use’’ (E. R. Q. A. Chesson, ed.), pp. 50–57. Elsevier Applied Science, London. Xiong, Y., Contento, A., and Bassham, D. (2005). AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana. Plant J. 42, 535–546. Xiong, Y., Contento, A., Nguyen, P., and Bassham, D. (2007). Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol. 143, 291–299. Yano, K., Suzuki, T., and Moriyasu, Y. (2007). Constitutive autophagy in plant root cells. Autophagy 3, 360–362. Yoshimoto, K., Hanaoka, H., Sato, S., Kato, T., Tabata, S., Noda, T., and Ohsumi, Y. (2004). Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy. Plant Cell. 16, 2967–2983. Zhang, X., Henriques, R., Lin, S., Niu, Q., and Chua, N. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646.
T H I R T Y- O N E
Chimeric Fluorescent Fusion Proteins to Monitor Autophagy in Plants Ken Matsuoka* Contents 1. Introduction 2. Fluorescent Proteins and Autofluorescence in Plant Cells and Organelles 3. Visual Detection of Autophagosomes and Autophagic Bodies Using Fluorescent Protein-Tagged ATG8 3.1. General note for the visual detection of autophagosomes in tobacco BY-2 cells expressing YFP-NtAtg8a under nutrient starvation conditions 3.2. Preparation of culture medium and standard culture conditions 3.3. Preparation of sugar-starved medium 3.4. Preparation of phosphate or nitrogen-starved medium 3.5. Nutrient starvation and detection of autophagosomes in living cells 3.6. Colocalization analysis of autophagosomes and other cellular structures 4. Visual Detection of Autophagic Degradation Using Fluorescent Protein-Tagged Synthetic Cargo 4.1. Aggregated fluorescent protein as a reporter protein 4.2. Expression and detection of autophagic degradation in tobacco BY-2 cells 4.3. Expression and detection of autophagic degradation in leaves of Arabidopsis plants expressing the cytochrome b5-DsRed fusion protein 5. Quantification of Fluorescent Fusion Proteins After Separation by Gel Electrophoresis
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5.1. Protein sample preparation, separation by SDS-PAGE, and detection of the DsRed fusion protein from transformed tobacco BY-2 cells Acknowledgments References
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Abstract Autophagy is induced under nutrient-deficient conditions in both growing tobacco BY-2 cultured cells as well as Arabidopsis and others intact plants. The fluorescent protein-tagged structural protein for autophagosomes, the Atg8 protein, allows nondestructive detection of autophagy induction in plant cells and tissues by fluorescence microscopy. Using this technique, the general operation of autophagy in growing root cells has been observed. A synthetic cargo protein for autophagy consisting of cytochrome b5 and the red fluorescence protein, DsRed, allows for the quantitative assay of autophagy in tobacco cells. This chapter describes methods for detecting autophagy in these plant cells using fluorescent protein fusions in situ with light microscopy, as well as quantification of autophagy.
1. Introduction Real-time detection of autophagic events is important for the molecular dissection of the process of autophagy. Fluorescent proteins, such as green fluorescent protein (GFP), red fluorescent protein (RFP), and derivatives of these proteins are now widely used to detect intracellular transport events. Yet special caution is necessary to use such fluorescent proteins in differentiated plant cells, including photosynthetic cells in green leaves and cells accumulating secondary metabolites; these cells have compounds that are highly fluorescent and tend to interfere with the detection of fluorescent proteins. The detection of autophagy induction can be done by monitoring the presence of autophagosomes in cells. The yeast Atg8 protein and its orthologs in higher organisms are widely used to detect autophagosomes, after tagging this protein with GFP and its derivatives (see, e.g., Mizushima and Kuma, 2008). Using this technique in combination with other methods, crucial roles for plant autophagy have been uncovered. These include degradation of oxidized proteins during oxidative stress (Sla´vikov et al., 2005; Xiong et al., 2007), disposal of protein aggregates (Toyooka et al., 2006), and possibly removal of damaged proteins and organelles during normal growth conditions as a housekeeping function (Sla´vikov et al., 2005; Yano et al., 2007). Quantification of autophagic degradation is also important for the elucidation of the autophagic process and its regulation. In yeast cells, activation of an alkaline phosphatase precursor, Pho8D60, dependent on the activity of
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vacuolar proteases was used to quantify autophagy and to isolate mutants in this pathway (Noda et al, 1995; see the chapter by Noda and Klionsky in this volume). In plants, we have developed a system to use fluorescent artificial cargo proteins to quantify autophagy in plant cells (Toyooka et al., 2006). In this chapter, I summarize the recent use of fluorescent proteins for the analysis of autophagy in plant cells and provide protocols and notes on these methods.
2. Fluorescent Proteins and Autofluorescence in Plant Cells and Organelles Developed plant cells tend to have a large quantity of fluorescence compounds, such as chlorophyll and lignin, which tend to interfere with the detection of fluorescent proteins. Chlorophyll is an essential pigment for photosynthesis that produces the green color in leaves. Although this compound is only found in chloroplasts, the volume of this organelle in photosynthetic cells is quite large, and accordingly this affects the analysis of fluorescent proteins in plant photosynthetic cells. In addition, chloroplasts in living cells change intracellular location and angles when cells are illuminated with visible light (Wada M et al., 2003). For example, when photosynthetic cells are illuminated with white light when adjusting the focus of the microscope, the chloroplasts tend to alter their orientation so that they face the plane of the light source to absorb the maximum intensity of light. Such behavior of chloroplast as well as the nature of chlorophyll, which emits red fluorescence with broad excitation wavelengths, tends to prevent the detection of intracellular structures when using a fluorescence microscope. The bottom wavelength of the excitation spectrum is approximately 480 nm (Fig. 31.1). This wavelength is similar to the excitation peak wavelength of GFP and yellow fluorescent protein (YFP). Thus, these proteins can be used without significant loss of detection sensitivity (see Fig. 31.1). Likewise, the red fluorescent protein DsRed has three-excitation peak wavelengths and one of the shortest is approximately 480 nm (Campbell et al., 2002). Thus, this protein can also be used with appropriate filter sets without interfering with the fluorescence of chlorophylls. The more problematic cells and tissues are lignified tissues and cells accumulating phenolic compounds. Lignin and related compounds have a high intensity of fluorescence with a broad excitation and emission spectrum (Willemse, 1989). Induction of a stress response induces formation of lignin and related compounds in plants. For example, application of the stress-related phytohormone methyl-jasmonate to tobacco BY-2 cells, which usually display very little fluorescence, induces the synthesis of phenolic compounds and the accumulation of a large quantity of fluorescent
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Relative intensity
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Figure 31.1 Relative intensity of excitation spectra of rice leaf at 700-nm emisson (gray line), EGFP (thick line) and DsRed (thin line).
pigments in vacuoles (Galis et al., 2006; Matsuoka and Galis, 2006). In this case, fluorescent proteins cannot be used to monitor cellular events because of the intense fluorescence of the central vacuoles, which comprise more than 90% (w/v) of the cell volume. Therefore, caution with regard to autofluorescence is needed to monitor autophagy in plant cells, as vacuoles are the lytic organelle for autophagic degradation in plants (Bassham 2007).
3. Visual Detection of Autophagosomes and Autophagic Bodies Using Fluorescent Protein-Tagged ATG8 Plants generally have multiple Atg8 proteins. Arabidopsis have nine Atg8 genes that are expressed differentially in different organs (Slavikova et al., 2005; Yoshimoto et al., 2002), and tobacco BY-2 cells express at least five distinct Atg8 orthologs (Toyooka and Matsuoka, 2006). Cellular functions and biochemical evens of these proteins as well as many of the homologs of other Atg proteins in plants during autophagy progression are similar to those observed in yeast and mammals (Doelling et al., 2002; Fujioka et al., 2008; Ketelaar et al., 2004; Phillips et al., 2008; Su et al., 2006; Thompson et al., 2005; Xiong et al., 2005). Thus, these proteins function as structural proteins for the autophagosome generation and can be used to detect autophagosomes and autophagic transport to the vacuoles in tobacco BY-2 cells, Arabidopsis protoplasts or Arabidopsis root cells after tagging GFP or YFP at their N terminus (Contento et al., 2005; Toyooka et al., 2006; Toyooka and Matsuoka, 2006; Yano et al., 2007; Yoshimoto et al., 2004). Essentially no difference in autophagosome detection was observed using different types of fluorescent protein tags in both Arabidopsis root cells and tobacco BY-2 cells under starvation conditions (Toyooka and Matsuoka, 2006;
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Yoshimoto et al., 2004). Thus, chimeric proteins consisting of one of the Atg8 family members and GFP or YFP expressed under the control of a constitutive promoter can be used to monitor autophagy by detecting the fluorescence of such proteins. The following are the protocols for monitoring autophagy induction in transformed tobacco cells that are easy to use for the detection of autophagosomes within a day of nutrient starvation.
3.1. General note for the visual detection of autophagosomes in tobacco BY-2 cells expressing YFP-NtAtg8a under nutrient starvation conditions Stably transformed tobacco BY-2 cells are subcultured weekly in 95 ml of culture medium as described previously (Toyooka et al., 2006). As cells reach the stationary phase of growth within a week due to the limitation of phosphate and nitrogen sources in the medium, autophagy is already induced in cells at such a growth phase (Toyooka et al., 2006). Thus, cells in logarithmic growth phase (i.e., 3 days after subculture) should be used in all experiments.
3.2. Preparation of culture medium and standard culture conditions 1. Dissolve 30 g of sucrose and the premixed powder Murashige & Skoog salts (Wako Pure Chemicals, Osaka, Japan) in approximately 900 ml of water. 2. Add 10 ml of 20 g/l KH2PO4 (pH not adjusted). 3. Add 1 ml of 1000x vitamins (1000x ¼ 100 g/l myo-inositol, 1 g/l thiamin-HCl). 4. Add 20 ml of 2,4-D stock (10 mg/ml 2,4-dichlorophenoxyacetic acid in ethanol). 5. Adjust the pH of the solution to 5.8 with 1 M KOH, and bring the volume up to 1000 ml. 6. Pour 95 ml into a 300-ml conical flask, and cover the opening with two layers of aluminum foil. 7. Autoclave at 121 C for 15 min, cool down to room temperature, and store in the dark. 8. Once a week, transfer 1.5 ml of culture at the stationary phase to 95 ml of fresh medium, and culture at 26.5 C 1 C with rotation of 130 rpm. In some case transformed cells grow slower than nontransformed cells. In this case, a larger volume of culture (up to 5 ml) is transferred to fresh medium.
3.3. Preparation of sugar-starved medium Use 15 g of mannitol instead of 30g of sucrose. The other conditions are the same as for the standard media described previously.
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3.4. Preparation of phosphate or nitrogen-starved medium
1. 1000x B, Mo solution: 6 g/l H3BO4, 250 mg/l Na2MoO42H2O. 2. 100x Fe, Mn, Zn solution: 1.7 g/l MnSO47H2O, 1.05 g/l ZnSO4 7H2O, 5.125 g/l FeSO47H2O, 3.75 g/l Na2EDTA. 3. 1000x trace elements: 830 mg/l KI, 25 mg/l CoCl26H2O, 25 mg/l CuSO45H2O. 4. 20x phosphate-free major salts: 33 g/l NH4NO3, 38 g/l KNO3, 8.8 g/l CaCl22H2O, 7.4 g/l MgSO47H2O. 5. 20x nitrogen-free major salts: 3.4 g/l KH2PO4, 8.8 g/l CaCl22H2O, 7.4 g/l MgSO47H2O. 6. Mix approximately 900 ml of water, 50 ml of appropriate major salts (either phosphate- or nitrogen-free), 1 ml of B, Mo solution, 10 ml of Fe, Mn, Zn solution, 1 ml of trace elements, 30 g of sucrose, 1ml of 1000x vitamins, and 20 ml of 2,4-D stock, and adjust to pH 5.8 with KOH. Sterilize as previously.
3.5. Nutrient starvation and detection of autophagosomes in living cells Transformed BY-2 cells expressing YFP-Atg8 at the logarithmic growth phase are transferred into nutrient-free MS medium to induce autophagy. 1. Place 10–50 ml of a 3-day-old culture into a 50-ml conical tube. 2. Centrifuge at 100 g for 5 min to pellet cells. 3. Remove the supernatant fraction and add the original volume of medium lacking one of the nutrients. Suspend the cells with gentle shaking. 4. Pellet the cells again by centrifugation to wash out the original medium. 5. Remove the supernatant fraction, and then add the same volume of medium lacking one of the nutrients. Suspend the cells. 6. Transfer the cell suspension into a conical flask of appropriate volume. We use 100-ml flasks for 10–30 ml of culture, 200-ml flasks for 30–60 ml of culture, and 300-ml flasks for 60–95 ml of culture. 7. Shake flasks using a rotary shaker at 26.5 C at a rotation speed 130 rpm. 8. After 24 h, an aliquot of the cell suspension is placed on a slide glass, covered with cover glass and monitored for the presence of autophagosomes using an epifluorescence microscope or laser-scanning confocal microscope with GFP or FITC filter sets. Dotted structures of 100– 1000 nm in diameter, which are moving slowly in the cells, are the autophagosomes (Fig. 31.2). Note: The expression level of YFP-NtAtg8a in tobacco BY-2 cells is not very high. Therefore, when observing the cells with an epifluorescence microscope equipped with the usual mercury lamp, a high-sensitivity CCD camera is necessary to detect autophagosomes. We routinely use an
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Figure 31.2 Autophagosome formation in transformed tobacco BY-2 cells expressing YFP-NtAtg8a. Logarithmic phase tobacco BY-2 cells expressing theYFP-NtAtg8a construct under the control of the CaMV35S promoter (left) and 24 h after induction of autophagy (right). Images were collected using an Olympus IX70 inverted fluorescence microscope with NBA filter sets with a Roper CoolSNAP HQ camera.The dotted structures in the left images are the autophagosomes.
Olympus IX70 or IX81 fluorescence microscope equipped with a Roper CoolSNAP HQ camera. Autophagy inhibitors 3-methyladenine or E-64 can be included during the incubation for a negative control. For details, see Takatsuka et al. (2004) or the chapter by Moriyasu and Inoue in this volume.
3.6. Colocalization analysis of autophagosomes and other cellular structures Colocalizaton studies of autophagosomes and other cellular structures are important for the analysis of autophagy targets under various induction conditions. However, as autophagosomes are transient structures during the entire autophagic degradation process, it is difficult to quantify the association of autophagosomes and cellular structures in living cells. Therefore we use acetone-fixed cells retaining YFP fluorescence (or YFP fluorescence and other fluorescent proteins in cells expressing two different fluorescent fusion proteins) to analyze the colocalization. 1. Cool 100% acetone to 20 C in a freezer. 2. Transfer 2 ml of autophagy-induced BY-2 cell culture into a 15-ml conical tube. 3. Centrifuge at 1000 g for 5 min. Remove culture medium and discard. 4. Suspend cells in 5 ml of PBS (10 mM sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.5). 5. Centrifuge as previously and remove as much PBS as possible. 6. Add 5 ml of cold acetone, mix, and keep in a freezer for 10 min. During this incubation most of the cells sediment to the bottom. 7. Remove acetone using a Pasteur pipette. 8. Wash cells by adding 10 ml of PBS, suspend, and collect the cells by centrifugation.
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9. Repeat PBS wash twice. 10. Mount cells on slides and analyze the colocalization of the YFP signal and DsRed or other fluorescent protein signals using a fluorescence microscope. Alternatively, fixed cells are further stained with antibodies as described previously to localize organelles (Toyooka et al., 2006).
4. Visual Detection of Autophagic Degradation Using Fluorescent Protein-Tagged Synthetic Cargo Detection of autophagosomes using fluorescent protein-tagged Atg8 is an easy and reproducible method for the detection of autophagy. Fluorescent autophagosomes tagged with Atg8 fusion proteins are seen as dots. Yet Atg8 and its orthologs tend to also form intracellular dot structures that are not related to autophagy (Kuma et al., 2007). Therefore, an alternative method is needed to confirm that autophagy is actually taking place. One of the systems to detect autophagic degradation is to monitor the relocation of cargo protein from the cytoplasm to the vacuole. As plant vacuoles occupy most of the cell volume, relocation of cytoplasmic protein to the vacuoles can be easily monitored using light microscopy. This strategy was used to detect transport of GFP-Atg8 from the cytoplasm to the vacuole in Arabidopsis root cells (Yoshimoto et al., 2004), as well as to monitor transport of a synthetic autophagic cargo from the cytoplasm to the vacuole in tobacco BY-2 cells (Toyooka et al., 2006). In this section, detailed information for the use of synthetic fluorescent cargo proteins to monitor autophagic degradation is discussed.
4.1. Aggregated fluorescent protein as a reporter protein Protein aggregates can be easily formed when a protein contains two or more oligomerization domains. Overexpression of such proteins tends to cause aggregates to form in the cell. One such protein is a fusion protein of cytochrome b5 and the original DsRed protein (Toyooka et al., 2006). Cytochrome b5 is an integral endoplasmic reticulum membrane protein, which tend to form into an octamer after solublization (Calabro et al., 1976). DsRed is a tetrameric red fluorescent protein (Baird et al., 2000). Thus, the expression of a fusion construct of these two proteins causes the self-association of a protein with two different affinity sites, namely from both cytochrome b5 and DsRed, and generates protein aggregates that emit strong red fluorescence after excitation with green light in tobacco BY-2 cells (Toyooka et al., 2006). The tetrameric nature of DsRed is essential for the generation of the aggregates, as a fusion protein of cytochrome b5
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and a monomeric mutant of DsRed causes the exclusive targeting of this fusion protein to the endoplasmic reticulum (supplemental figure in Toyooka et al., 2006).
4.2. Expression and detection of autophagic degradation in tobacco BY-2 cells 1. Generate a cytochrome b5-DsRed fusion protein construct (Toyooka et al., 2006) under the control of a strong promoter, such as the tandem 35S promoter (Matsuoka and Nakamura, 1991) and introduce into tobacco BY-2 cells. 2. Culture stably transformed cells as described above (Preparation of culture medium and standard culture conditions) and observe red fluorescence using a fluorescence microscope with a RFP filter set. 3. At log phase, most of the cells contain punctate protein aggregates with red fluorescence (Fig. 31.3). 4. Induce autophagy as previously using sugar-, phosphate- or nitrogenfree media. Red fluorescence can be seen emitting from most parts of the cells, indicating that some of the RFP fusion protein is targeted to the vacuole and converted to a soluble form (see Fig. 31.3). 5. If required, vacuolar membrane can be stained with a styryl dye, FM 1-43 (Invitrogen, T35356), which gives green fluorescence under blue light excitation (Emans et al., 2002). In this case, an aliquot of stock solution of FM 1-43 in DMSO (20 mM) was mixed with the autophagy-induced culture to a final concentration of 20 mM, and further incubated for 2 h at 26.5 C with shaking.
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Figure 31.3 Expression and aggregate formation of cytochrome b5-DsRed fusion protein in tobacco BY-2 cells and vacuolar targeting of the cytochrome b5-DsRed fusion protein into vacuoles. Logarithmic phase tobacco BY-2 cells expressing a cytochrome b5-DsRed construct under an enhancer duplicated derivative of the CaMV35S promoter (left) and 24 h after the induction of autophagy (right). Images were collected using an Olympus IX70 inverted fluorescence microscope withWIG filter sets equipped with an Olympus DP70 digital camera.
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6. Likewise, the lumen of the vacuoles can be stained with BCECF to emit green fluorescence (Matsuoka et al., 1997). In this case, an aliquot of stock solution of BCECF-AM in DMSO (Invitrogen, B3051) was mixed with the autophagy-induced culture to a final concentration of 6 mM, incubated for 5 min at room temperature and washed the cells in the culture with flesh medium. Fluorescence of BCECF was monitored using fluorescence microscopes with a standard GFP filter setting. Note: Roots of transformed Arabidopsis plants can be analyzed with the same setting of the microscope.
4.3. Expression and detection of autophagic degradation in leaves of Arabidopsis plants expressing the cytochrome b5-DsRed fusion protein Green leaves contain a lot of chlorophyll and other pigments for photosynthesis. Thus, choosing the proper wavelengths of both excitation and fluorescence emission is necessary to obtain clear images of RFP fusion proteins. 1. Transform Arabidopsis plants using a dipping method (Zhang et al., 2006) and collect seeds from kanamycin-resistant T1 plants. 2. Germinate kanamycin-resistant seeds on Murashige-Skoog plate as described (Zhang et al., 2006). 3. Observe fluorescence of DsRed in leaves using a Zeiss LSM 510 META or other confocal laser scanning microscope with variable absorption wavelength. Choose 488-nm laser excitation and 580-nm fluorescence emission. Chloroplasts show weak fluorescence at 580 nm under blue light excitation (Terao et al., 1995). Thus, this condition allows the detection of fluorescent aggregates in the presence of weak background fluorescence from chloroplasts in the leaves of transformants, whereas no such structures were seen in control plants without expressing cytochrome b5-DsRed fusion protein (Fig. 31.4). 4. Detach leaves from seedlings and incubate with water for 24 h in the dark to induce autophagy. Under this condition a decrease in the red fluorescent puncta is observed. For control, leaves are incubated with Murashige-Skoog medium containing 1% (w/v) sucrose. Note: Recording of DsRed fluorescence in roots can also be done with the same condition as for leaves. The emission fingerprinting protocol of the Zeiss LSM 510 META microscope allows more clear separation of chloroplasts and aggregates. However, patterns of chloroplast fluorescence change under growth and starvation conditions. Thus, as a control, the fluorescence patterns of chloroplasts should be determined experimentally using nontransformed Arabidopsis leaves grown or incubated exactly under the same condition as for leaves expressing the cytochrome b5-DsRed fusion protein.
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Figure 31.4 Expression and aggregate formation of a cytochrome b5-DsRed fusion protein in Arabidopsis plants. Upper panels show transgenic and control plants. Middle panels show red fluorescence emitted from Arabidopsis leaves after excitation with a 488-nm laser and 580-nm florescence emission using a Zeiss LSM 510 META microscope. Punctate signals of the aggregates of the cytochrome b5-DsRed fusion protein are apparent in the transformants expressing the cytochrome b5-DsRed fusion protein. Lower panels show red fluorescence emitted from Arabidopsis roots. Arrowheads in middle and lower panels indicate some of the aggregates of the cytochrome b5-DsRed fusion protein.
5. Quantification of Fluorescent Fusion Proteins After Separation by Gel Electrophoresis It is not sufficient to monitor autophagy by fluorescence microscopy; it is also necessary to quantify autophagic activity for the full molecular dissection of autophagy. Here, I describe a method to quantify autophagic degradation using the chimeric fusion proteins described previously. A basic background on the quantification of autophagy is that, when proteins are transported to a lytic environment, such as the lumen of the vacuole, these proteins are degraded partially or completely by proteases in the organelle. The relatively stable nature of the fluorochromes of DsRed and GFP in the presence of SDS allows us to detect the fusion proteins or derivatives after separation by SDS-polyacrylamide gel electrophoresis (Baird et al., 2000; Shimizu et al., 2005). Thus, vacuolar delivery of
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cytochrome b5-DsRed and conversion of this protein to a soluble form through proteolytic processing by vacuolar proteases can be detected as the change of relative molecular mass of the RFP in transformed plant cells expressing the fusion construct.
5.1. Protein sample preparation, separation by SDS-PAGE, and detection of the DsRed fusion protein from transformed tobacco BY-2 cells 1. Culture stably transformed tobacco BY-2 cells expressing the fusion protein as previously and induce autophagy. 2. Transfer 1 ml of cells into a 15-ml round-bottomed polypropylene tube (e.g., Falcon #352063). 3. Spin the tube at 1000 g for 5 min. 4. Collect and measure the volume of the supernatant fraction. 5. Add 1 ml of PBS (pH 7.5 at room temperature) to the cell pellet. Suspend by gentle shaking. 6. Centrifuge as previously, remove supernatant fraction, and discard. 7. Add 1 ml of PBS to cell pellet and suspend cells. Chill on ice for at least 5 min (up to 1 h without any effect on the processing). 8. Sonicate the cell suspension using a probe-type sonicator (UR-20P, TOMY Seiko, Tokyo, Japan) for 30 s, twice with a 1-min interval between sonications. 9. Transfer the disrupted cell suspension into a 1.5-ml microcentrifuge tube and centrifuge at 1000 g for 5 min at 4 C. 10. Collect the supernatant fraction. The supernatant fraction may be stored at 20 C at this point if required. 11. Mix the supernatant fraction (disrupted cells) and an equal volume of 2x SDS-PAGE loading buffer [0.01% (w/v) bromophenol blue, 4% (w/v) SDS, 20% (w/v) glycerol, 100 mM Tris-HCl, pH 8.8]. 12. Incubate at room temperature for 10–30 min and apply to a 12.5% (w/v) polyacrylamide minigel of 0.75 mm thickness using the Hoefer SE 260 Mighty Small system or equivalent. 13. Apply 25 mA current per gel until the dye front reaches the bottom of the gel. 14. After the separation of proteins, the gel is separated from the plates and placed into a plastic bag. 15. Put the plastic bag onto the glass stage of a Typhoon 8600 or 9400 image scanner (GE Health Care, London). 16. Scan the gel image using a 532-nm excitation laser at 650V through a 580BP30 emission filter, which allows the detection of DsRed. A typical image of the detection of autophagic degradation of the cytochrome b5-DsRed fusion protein is shown in Fig. 31.5.
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Figure 31.5 Processing of cytochrome b5-DsRed fusion protein under starvation conditions. Rapidly growing transformed tobacco BY-2 cells expressing the cytochrome b5-DsRed fusion protein were incubated for 24 or 48 h in complete culture medium or medium lacking either sucrose, nitrogen containing compounds or phosphate. Proteins prepared from cells were separated by SDS-PAGE, and DsRed-related proteins were detected using a Typhoon 8600 image analyzer. Open arrowhead indicates intact cytochrome b5-DsRed protein. Closed arrowhead indicates processed cytochrome b5-DsRed protein accumulated in the vacuoles.
Note: Prolonged incubation of protein samples with sample buffer or heating samples abolishes the fluorescence. The same quantification procedure can be applied for transformed Arabidopsis leaves and roots. In this case tissues are homogenized with the same buffer using a motor and pestle. After scanning, gels can either be stained with an appropriate dye to detect proteins or can be used for immunoblotting. Fluorescence of GFP or YFP fusion proteins can also be detected using the same scanners with different settings (Shimizu et al., 2005). As GFP or YFP fluorochromes are unstable in acidic conditions, fusions with these proteins and Atg8 tend to lose fluorescence after targeting to vacuoles.
ACKNOWLEDGMENTS I thank Drs. Kiminori Toyooka in the RIKEN Plant Science Center and Ivan Galis in the Max Planck Institute for Chemical Biology for sharing data presented in this work. This work was supported in part by a grant-in-aid for scientific research from MEXT Japan.
REFERENCES Baird, G., Zacharias, D., and Tsien, R. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989. Bassham, D. (2007). Plant autophagy: More than a starvation response. Curr. Opin. Plant Biol. 10, 587–593. Calabro, M., Katz, J., and Holloway, P. (1976). Self-association of cytochrome b5 in aqueous solution. Gel filtration and ultracentirfugational studies. J. Biol. Chem. 251, 2113–2118.
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