Article |
Address correspondence to Masayuki Miura, Laboratory for Cell Recovery Mechanisms, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel.: 81-48-467-6945. Fax: 81-48-467-6946. E-mail: miura{at}brain.riken.go.jp
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Abstract |
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Key Words: FRET; caspase-3; caspase-9; nuclear activation of caspase-3; apoptosis
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Introduction |
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Fluorescence resonance energy transfer (FRET)* technology can provide significant information about the dynamics and pattern of endogenous caspase activation in vivo. Several papers have reported the use of recombinant caspase substrates composed of enhanced cyan fluorescence protein (ECFP) as the FRET donor and enhanced yellow fluorescence protein (EYFP) as the FRET acceptor, linked by peptides containing the caspase-3 cleavage sequence, DEVD (Tyas et al., 2000; Luo et al., 2001; Rehm et al., 2002). The development of such indicators has enabled researchers to monitor caspase activation at the single-cell level in real time. However, these indicators may have some limitations in the in vivo monitoring of caspase activation for a number of reasons. First, the molar extinction coefficient of the acceptor protein is an important factor for determining the efficiency of FRET; however, the molar extinction coefficient of EYFP is highly dependent on the proton (H+) concentration, especially under physiological pH conditions (pH 7.3; Llopis et al., 1998). Second, EYFP is also highly sensitive to chloride ions (Cl-; Jayaraman et al., 2000). In fact, owing to these characteristics, EYFP has been used as an indicator for pH and Cl-; EYFP-expressing transgenic mice are successfully used to detect pH and Cl- concentration changes in vivo (Metzger et al., 2002). Thus, with changes in H+ or Cl- concentrations, indicators using EYFP as the FRET acceptor may detect signals that are not caused by caspase activity. Several reports have suggested that acidification occurs during apoptosis (Perez et al., 1995; Vincent et al., 1999; Matsuyama et al., 2000) and that the Cl-/HCO3- antitransporter is involved in apoptotic events (Maeno et al., 2000; Araki et al., 2002). Because a change in Cl- concentration is also known to be associated with neuronal activity, it is crucial to use a Cl- insensitive indicator for monitoring caspases in neurons. A ratiometric indicator (Clomeleon) in which CFP combined with YFP via a flexible peptide linker, similar to the CY3 construct, was used as a reporter of intracellular Cl- concentration ([Cl-]i) in cultured hippocampal neurons (Kuner and Augustine, 2000). Using Clomeleon, changes in the somatic [Cl-]i were observed to spread into dendrites after the focal activation of GABAA receptors on the soma of a neuron. It would be hard to distinguish whether changes in the ECFP/EYFP ratio were caused by [Cl-]i changes or caspase activation in neurons. For these reasons, EYFP-based activated caspase indicators must be improved for the reliable monitoring of caspase activation in vivo.
Venus is a variant of EYFP that exhibits fast and efficient maturation at 37°C (Nagai et al., 2002). The use of Venus as an acceptor for FRET in the Ca2+ indicator (YC2.12) allows the Ca2+ signal to be detected immediately after the gene is introduce into cells. Furthermore, the absorption efficiency of Venus is significantly less sensitive to H+ and Cl- than that of EYFP. Therefore, Venus should be a more suitable FRET acceptor than EYFP in indicators for activated caspase. In this paper, we report an improved indicator for caspase activation based on the FRET method (SCAT) that uses Venus in place of EYFP. We clearly show that SCAT is highly resistant to changes in H+ and Cl- concentration in vitro and insensitive to environmental effects in living cells, allowing us to detect reliable signals for caspase activation. We also show the spatio-temporal dynamics of caspase-3 (DEVDase) activation in the cytosol and nuclei using SCAT3. Finally, by monitoring caspase-9 (LEHDase) in living HeLa cells using SCAT9, we report for the first time a difference in the spatio-temporal dynamics between initiator and executioner caspases.
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Results |
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The value for the 530/475 nm emission ratio of SCAT3 changed from 2.29 to 0.90 in response to TNF-/CHX treatment, whereas the ratio for CY3 changed from 1.86 to 0.68 (Fig. 1 D). The wider dynamic range of the FRET signal from SCAT3 (1.40 ± 0.08; n = 3) compared with that from CY3 (1.18 ± 0.05; n = 3) may be explained by the complete maturation of Venus, the acceptor contained in SCAT3.
SCAT is resistant to pH and Cl- concentration changes
Our initial goal was to generate an indicator for caspase activation under physiological conditions, such as in a developing embryo. It is important that the indicator does not detect any signals except for caspase activation. We noticed that CY3 was significantly sensitive to changes in the concentration of both H+ and Cl- (Fig. 2, A and B) in vitro. For example, acidification from pH 7.5 to 7.0 decreased the emission ratio of CY3 by 26%. Under the same conditions, only a 0.01% decrease was observed for SCAT3. Therefore, SCAT3 was highly stable under these conditions compared with CY3.
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Specificity of SCAT3 for activated caspase-3 in apoptotic HeLa cell extract
Next, we examined the specificity of SCAT3 for activated caspases in vitro. In vitro-synthesized SCAT3 protein was incubated with several activated caspases (Fig. 3 A). It was efficiently cleaved by caspase-3 and partially cleaved by caspase-8 and caspase-9 in vitro. In contrast, little SCAT3 was cleaved by caspase-6. To investigate the specificity of SCAT3 in the TNF treated cell lysates, we performed immunodepletion experiments to remove caspase-3 from TNF-/CHX-treated HeLa cell lysates. Caspase-3 activation was observed at TNF-
/CHX-treated lysates (Fig. 3 B), then caspase-3 precursor and its activated form were depleted from apoptotic lysates by an anti-caspase-3 antibody. As a control, we incubated TNF-
/CHX-treated lysates with an anti-GFP antibody. We then examined SCAT3 cleavage in these extracts. SCAT3 was effectively cleaved in the control TNF-
/CHX-treated lysates (whether or not they had been treated with the GFP antibody). On the other hand, only a small portion of the SCAT3 was cleaved in the caspase-3depleted lysates. These results indicated that most of the SCAT3 cleavage in TNF-
/CHX-treated HeLa cell lysates was done by caspase-3.
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Discussion |
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We showed another application of SCAT by creating SCAT9, which contained LEHD in its linker sequence. The specificity of this indicator for caspase-9 was examined by an in vitro cleavage assay and immunodepletion assay using dATP/cytochrome cactivated apoptotic HeLa cell lysates. The in vitro cleavage assay revealed that SCAT9 was cleaved not only by caspase-9, but also by caspase-8. If caspase-8 is strongly activated in cells, SCAT9 should detect the activation of caspase-8 as well. In apoptotic HeLa cell lysates, SCAT9 cleavage was significantly suppressed by the depletion of caspase-9, indicating that SCAT9 is mainly cleaved by caspase-9 in these samples. Because we could detect partial cleavage of SCAT9 in the caspase-9depleted apoptotic lysates, the possible involvement of other caspases cannot be excluded. Another possibility is that residual caspase-9 in the lysates is involved in the cleavage of SCAT9 after caspase-9 immunodepletion. Because caspase-9 is incorporated into the apoptosome, which is a large protein complex, it might prevent the access of the caspase-9 antibody to completely deplete the caspase-9 from the apoptotic lysates.
The caspase-9 activation profile was significantly different from that of caspase-3. The changes in the emission ratio of SCAT9 occurred very slowly compared with the changes in that of SCAT3. Caspase-3 activation was completed before the initiation of apoptotic morphological changes, but caspase-9 activation continued to progress after the morphological changes. These different rates of caspase-3 and caspase-9 activation may be caused by differences in their potential protease activity. When we measured the enzymatic activity of caspase-9 (LEHDase activity) and caspase-3 (DEVDase) in TNF-/CHX-stimulated HeLa cells, the DEVDase activity was six times higher than that of LEHDase. Caspase-9 would thus take longer to complete the cleavage of SCAT9 than caspase-3 takes to cleave SCAT3 if there are similar levels of the SCAT substrates in cells. In spite of the differences in activation profiles between caspase-3 and -9, the timing of the initiation of these caspases was almost the same (Fig. 7 D). Thus, we provide the first and direct evidence that activation of the initiator caspase and the executioner caspase is temporally coupled to initiate apoptotic changes.
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Materials and methods |
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Cell culture and transfection
HeLa cells were maintained in DME (Sigma-Aldrich) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS. Transfections of HeLa cells were performed using the SuperFect reagent (QIAGEN).
Spectral analysis
SCAT3-expressing HeLa cells were exposed to 10 µg/ml CHX or 50 ng/ml TNF-/10 µg/ml CHX for 6 h, then washed three times in PBS at 37°C. The cells were resuspended in PBS and used for fluorescence spectra determinations with an excitation wavelength of 435 nm at 37°C using a fluorescence spectrophotometer (model F2500; Hitachi).
pH and Cl- titration analysis
For pH titration, a series of buffers was prepared with pHs ranging from 6 to 9 in either 50 mM 2-(N-morpholino) ethanesulfonate, Hepes, or Tris, in the presence of 0.5% Triton X-100, 100 mM KCl, and protease inhibitors (5 µg/ml pepstatin, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 0.1 mM PMSF). For Cl- titration, a series of buffers was prepared with Cl- concentrations ranging from 0 to 450 mM in 0.5% Triton X-100 containing 50 mM Hepes-KOH, pH 7.4, 5 µg/ml pepstatin, 10 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.1 mM PMSF. The ionic strength of these solutions was adjusted to 450 mM with potassium D-gluconate.
HeLa cells (2 x 105 cells) were plated on 6-well plates and harvested at 18 h. The cells were transfected with 1 µg pSCAT3 or pCFP-DEVD-YFP (CY3). 24 h after transfection, SCAT3- or CY3-expressing HeLa cells were washed with PBS (37°C) and incubated in the indicated solutions at 37°C for 10 min. The supernatant, obtained by centrifugation at 10,000 g for 10 min at 37°C, was subjected to measurement of the 530/475-nm emission ratio with an excitation wavelength of 435 nm at 37°C.
Western blotting
Cell lysates were prepared by washing cells in PBS, then resuspending the cell pellet in sample buffer (62.5 mM Tris-HCl, pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromophenol blue, and 5% mercaptoethanol). The sample was immediately incubated for 10 min at 95°C, and the cell debris was pelleted before loading the sample onto an SDS-polyacrylamide gel (12%). After electrophoresis, the proteins were transferred onto an ImmobilonTM membrane (Millipore) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). An anti-myc mouse mAb (Invitrogen) and HRP-conjugated antimouse IgG (Promega) were used to detect full-length and cleaved SCAT.
Imaging analysis
Cells were plated on polyethyleneimine-coated glass coverslips and transfected with 0.5 µg plasmid vector for 6 h, then maintained in growth medium for another 12 h. During imaging, the cells were maintained in HBSS (GIBCO BRL) containing 10 mM Hepes, pH 7.4, and placed in a heated chamber. Imaging analysis was carried out using illumination pillars (ILL100LH; Olympus) with an interlined charge-coupled device camera (CoolSNAP HQTM; Roper Scientific) controlled by MetaFluor 4.6.5 Software (Universal Imaging Corp.). A 440AF21 excitation filter, a 455DRLP dichroic mirror, and two emission filters (480AF30 for ECFP and 535AF25 for Venus or EYFP) were used for SCAT or CY3 imaging. The two emission filters alternated with a filter changer (Lambda 10-2; Sutter Instrument Co.).
In vitro cleavage of SCAT3 and SCAT9
The in vitro cleavage assay was performed as described previously (Nakanishi et al., 2001). The SCAT3 or SCAT9 protein was prepared by in vitro translation and transcription using a TNT-coupled reticulocyte lysate system (Promega). 2 µl SCAT3 or SCAT9 protein was incubated at 37°C with 1 U purified, active caspase-3, -6, -8, or -9 (MBL International Corporation) for 1 h. The reaction mixture contained 20 mM Hepes-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 19 µg/ml aprotinin, and 0.1 mM PMSF. Western blot analysis with the anti-Myc antibody was used to detect the proteins, as described above.
Preparation of apoptotic extracts for SCAT3 and SCAT9 cleavage assay
For SCAT3 cleavage assay, HeLa cells were treated with 50 ng/ml TNF- and 10 µg/ml CHX and then cultured for 6 h. After washes by PBS two times, cells were suspended by digitonin buffer (10 µM digitonin, 50 µM Tris-HCl, pH 7.5, 1 mM EDTA, and 10 mM EGTA). Samples were incubated at 37°C for 10 min and then centrifuged at 15,000 rpm. The supernatant was collected and MgCl2 was added at final concentration of 2 mM.
For SCAT9 cleavage assay, HeLa cells were harvested by centrifugation at 1,000 rpm for 5 min. After washes in PBS and Buffer A (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin), the cells were suspended in two vol of buffer A and incubated 20 min on ice. The cells were then homogenized by 15 strokes with a Dounce homogenizer using a B-type pestle. The lysates were clarified by centrifugation at 10,000 g for 1 h. The supernatant was collected, and NaCl was added to a final concentration of 50 mM. The in vitro apoptotic reaction was carried out by the addition of 10 µM cytochrome c and 1 mM dATP for 2 h at 37°C in 3 µg/µl lysate before immunodepletion experiment.
Immunodepletion of caspase-3 or caspase-9 from apoptotic extracts
Aliquots of protein G Sepharose (40 µl) were precoated with 5 µg anti-caspase-9 rabbit pAb, provided by Drs. Yutaka Eguchi and Yoshihide Tsujimoto (Osaka University, Osaka, Japan; Eguchi et al., 1999), 5 µg anti-caspase-3 mouse mAb (Transduction Laboratories), or 5 µg anti-GFP rabbit pAb (MBL International Corporation) in a total volume of 300 µl in Buffer A, and were rocked for 3 h at 4°C. Antibody-coated beads were washed three times with Buffer A, then mixed with apoptotic HeLa cell lysates (150 µg of cytochrome c/dATP apoptotic lysate for SCAT9 and 100 µg of TNF-/CHX-treated apoptotic lysate for SCAT3 as described above) and rocked for 3 h at 4°C. The beads were removed from the lysates before SCAT3 or SCAT9 cleavage assay.
Online supplemental materials
Videos 1 and 2 show real-time imaging analyses of apoptotic HeLa cells using the SCAT3 and SCAT9 indicators, respectively. HeLa cells were treated with 50 ng/ml TNF- and 10 µg/ml CHX. Changes in the emission ratio are represented as red (high ratio) to blue (low ratio). Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200207111/DC1.
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Footnotes |
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* Abbreviations used in this paper: Cl-, chloride ion; [Cl-]i, intracellular Cl- concentration; CHX, cycloheximide; ECFP, enhanced cyan fluorescence protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; H+, proton; NLS, nuclear localization signal; STS, staurosporine; TNF-
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Acknowledgments |
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This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, Culture, and Technology to M. Miura. This work was also supported in part by grants from the RIKEN Bioarchitect Research Project. K. Takemoto is a research fellow of the Junior Research Associate Program, RIKEN.
Submitted: 19 July 2002
Revised: 9 December 2002
Accepted: 9 December 2002
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