2.1.

Gene Construction

For this study we constructed two different recombinant FRET probes, CFP-VDVAD-DsRed2 and CFP-DEVD-DsRed2; they were each made by fusing two fluorescent proteins (ECFP and DsRed2) with a linker containing the caspase-2 cleavage sequence^{10, 22} (VDVAD) and the caspase-3 cleavage sequence^{9, 11, 13} (DEVD), respectively. To construct CFP-VDVAD-DsRed2 (CD2) and CFP-DEVD-DsRed2 (CD3), the ECFP-EYFP concatemer from YC2.1 (a gift from A. Miyawaki) was subcloned into PMD18-T (CLONTECH) by HindIII and EcoRI. The enhanced mutants ECFP and EYFP are referred to using the more concise names CFP and YFP throughout. The HindIII∕SacI fragment from the PMD18 was replaced with the HindIII∕SacI-digested polymerase chain reaction (PCR) product. The primers for CD2 were as follows: forward, $5^{\prime }$ -ATAAGCTTGCGGCCGCCACCATGGTGAC- $3^{\prime }$ , reverse, $5^{\prime }$ -AGGAGCTCGGATCCATCTGCGACATCGACCTCTTCTGTCAGTTGGTCATGC- $3^{\prime }$ . The primers for CD3 were as follows: forward, $5^{\prime }$ -ATAAGCTTGCGGCCGCCACCATGGTGAC- $3^{\prime }$ , reverse, $5^{\prime }$ -AGGAGCTCACCACCATCGACCTCATCCTCTTCTGTCAGTTGGTCATGC- $3^{\prime }$ . According to this protocol, the VDVAD and DEVD sequences were introduced into the N terminal of CFP, respectively. Then, DsRed2 was amplified from pDsRed2 (CLONTECH) using the following primers: forward, $5^{\prime }$ -ACTAGAGCTCATGGCCTCCTCCGAGAACG- $3^{\prime }$ , reverse, $5^{\prime }$ -ACAGAATTCCTACAGGAACAGGTGGTGG- $3^{\prime }$ . The amplified fragment was digested and ligated into CFP-VDVAD-YFP∕PMD18-T using SacI and EcoRI, which replaced YFP with DsRed2. CFP-VDVAD-DsRed2 was also subcloned into pcDNA3 to generate pcDNA3-CFP-VDVAD-DsRed2, (CD2) which could express in the mammalian cells. PcDNA3-CFP-DEVD-DsRed2 (CD3) was generated by the same procedure.

2.2.

Expression, Purification, and Cleavage of these Recombinant FRET Probes in Vitro

To identify the sensitivity and specificity of CD2 and CD3 to caspase activation, the recombinant FRET probes (CD2 and CD3) were purified for in vitro analysis. CFP-VDVAD-DsRed2 and CFP-DEVD-DsRed2 were, respectively, subcloned to a prokaryotic fusion protein expression vector pET-28b and expressed in BL21 (DE3) Escherichia coli bacteria; these bacteria were cultured in Luria-Bertani (LB) at $37°\mathrm{C}$ and expression induced by $1\text{‐}\mathrm{mM}$ isopropyl-1-thio- $\beta \text{‐}D$ -galactopyranoside (IPTG) for $4\mathrm{h}$ at $30°\mathrm{C}$ when grown to $A_{600\mathrm{nm}}=1$ . Cells were harvested by centrifugation at $5000\times g$ for $10\min$ , washed in binding buffer ( $10\text{‐}\mathrm{mM}$ imidazole, $20\text{‐}\mathrm{mM}$ phosphate, $0.5\text{‐}\mathrm{M}$ NaCl), then resuspended in $20\mathrm{ml}$ lysis buffer, and, subsequently, were lysed by repeated cycles of freezing and thawing plus sonification. The recombinant protein was further purified by Ni-NTA resin (Qiagen).

Recombinant human active caspase-2 and caspase-3 were purchased from BioVision. One unit of recombinant human caspase was defined as the enzyme activity that cleaves $1\mathrm{nmol}$ of the individual caspase substrate (VDVAD-pNA for caspase-2, DEVD-pNA for caspase-3) per hour at $37°\mathrm{C}$ . Five micrograms of the purified recombinant protein were incubated with $1\text{unit}$ of each caspase in $10\mu \mathrm{l}$ of reaction buffer ( $50\text{‐}\mathrm{mM}$ NaCl, 0.1% CHAPS, $10\mathrm{mM}$ EDTA, 5% glycerol, and $10\text{‐}\mathrm{mM}$ dithiothreitol) at $37°\mathrm{C}$ for $1.5\mathrm{h}$ and then cleavage products of the FRET probes were analyzed by Western blot and spectroscopic measurements.

2.3.

Cell Culture and Gene Transfection

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with penicillin G sodium $(100\text{units}∕\mathrm{ml})$ , streptomycin sulfate $(100\mu \mathrm{g}∕\mathrm{ml})$ , and 10% fetal bovine serum at $37°\mathrm{C}$ in a humidified atmosphere of 5% $\mathrm{C}{\mathrm{O}}_2∕95\%$ air. Transfection of HeLa cells was performed using the GeneJammer® transfection reagent (Stratagene). Transfected cells were selected by exposure to Geneticin ( $800\mu \mathrm{g}∕\mathrm{ml}$ , Sigma), and stable cell lines were cloned. The properties of CD2 and CD3 were analyzed using CD2 or CD3 expressing HeLa cells by Western blot and spectroscopic measurements.

2.4.

Western Blotting

Purified fusion proteins were prepared for in vitro caspase assay as follows: the polyhistidine-tagged FRET sensor protein was expressed in bacteria and purified using a Ni-NTA affinity column. Then, the purified FRET probes CD2 or CD3 were incubated with caspase-2 or caspase-3 for $60\min$ at $37°\mathrm{C}$ . The samples were incubated for $10\min$ at $95°\mathrm{C}$ and then loaded for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Whole cell extracts were prepared as follows: HeLa cells that stably expressed CD2 or CD3 were collected at $800\mathrm{rpm}$ for $8\min$ and washed with phosphate-buffered saline (PBS), and then the cell pellet was resuspended in lysis buffer [ $62.5\text{‐}\mathrm{mM}$ Tris-HCl, pH 6.8, 10% (v∕v) glycerin, 2% (w∕v) SDS, $1\text{‐}\mathrm{mM}$ phenylmethylsulfonyl fluoride, $1\mu \mathrm{g}∕\mathrm{ml}$ pepstatin A, $1\mu \mathrm{g}∕\mathrm{ml}$ leupeptin, and $5\mu \mathrm{g}∕\mathrm{ml}$ aprotinin]. The cell lysates were immediately incubated for $10\min$ at $95°\mathrm{C}$ , the cell debris was pelleted, and the samples were loaded for SDS-PAGE.

Proteins were separated at $120\mathrm{V}$ for $1.5\mathrm{h}$ in SDS-PAGE (12%) and subsequently transferred onto a polyvinylidene difluoride membrane (PVDF, Amersham). Following blotting, the membranes were probed with anti-GFP antibody (1:3000) (Clontech) in triethanelamine-buffered saline (TBS) with 0.5% Tween 20. The immunoblots were then probed with horseradish-peroxidase-conjugated secondary goat anti-rabbit IgG (immunoglobulin G) antibody (1:5000) (Bio-Rad), and the bands were detected using the ECL Western-blotting analysis system (Amersham).

2.5.

Spectroscopic Measurements

The purified recombinant proteins (CD2 and CD3) were transferred into a quartz cuvette (total volume $1.5\mathrm{ml}$ ), and then incubated respectively with a $3\text{‐}\text{unit}$ recombinant human active caspase-2 or caspase-3 at $37°\mathrm{C}$ in the sample holder of the spectrofluorometer (LS-50B, Perkin-Elmer, Norwalk, Connecticut). Fluorescence emission spectra from $450\text{to}600\mathrm{nm}$ were recorded with an excitation wavelength at $433\mathrm{nm}$ .

The CD2 or CD3 expressing HeLa cells were each divided into two groups. One of the groups was treated by cisplatin for $8\mathrm{h}$ and the other was not. The cells were harvested, respectively, washed twice with PBS, resuspended in PBS (total volume $1\mathrm{ml}$ ), and then transferred into a quartz cuvette, which was placed inside a sample holder of the spectrofluorometer (Hitachi RF4500). The fluorescent emission spectrum was obtained by performing a spectrum scanning analysis with an excitation wavelength of $433\mathrm{nm}$ .

2.6.

Imaging Analysis System for FRET Measurements

FRET measurements were performed as described previously^{17, 22} on an inverted fluorescent microscope (Olympus IX70, Japan). FRET filter cubes for CFP∕DsRed2 were as follows: CFP (425-445HQ, DM450, 460-510HQ, Olympus); DsRed (BP510-550, DM570, BA590, Olympus); FRET ( $436∕20\mathrm{X}$ , 455DCLP, $620∕60\mathrm{M}$ , Omega Optical).

Images were captured using a cooled CCD camera (Micromax $5\mathrm{MHz}$ , Roper Scientific). Photographs were taken with Winview32 software (Roper Scientific). The ratio of FRET $\left(R_{\mathrm{FRET}}\right)$ was calculated by Matlab7.0 software as follows:²³

3.1.

Characterization of the FRET Probes (CD2 and CD3) for Activated Caspases

We generated two FRET probes by fusing a CFP and a DsRed2 protein with a specialized 17-amino-acid (CD2) or 16-amino-acid (CD3) linker. The linkers each contain the optimum caspase-2 cleavage sequence^{16, 22} (VDVAD) and caspase-3 cleavage sequence^{9, 11, 13} (DEVD), respectively (Fig. 1 ). Before caspases were activated, FRET from CFP to DsRed2 occurred because the two fluorescent proteins were covalently linked together. After caspases activation, cleavage of the linker peptide abolished the FRET (Fig. 1). To determine the sensitivity and specificity of CD2 and CD3 to activated caspases, we first examined these FRET probes for activated caspases in vitro. The recombinant FRET probes (CD2 and CD3) were expressed in BL21 (DE3) E. coli (Novagen), and purified by Ni-NTA resin (Qiagen). The changes of FRET efficiency in response to activated caspase cleavage were measured using a spectrofluorometer. The results are shown in Figs. 2a and 2b . After adding 3 units of activated caspases (BioVision) into the solution of CD2 or CD3, we observed a significant decrease of emission at the $580\text{‐}\mathrm{nm}$ wavelength during each $10\text{‐}\min$ scanning interval. The emission spectra normalized at the donor emission peak, enabling the acceptor emission to stand for the FRET donor∕acceptor ratio. The ratio value gradually decreased as time elapsed [Figs. 2a and 2b]. These results demonstrate that the CD2 and CD3 are sensitive to the activated caspase-2 and caspase-3, respectively. To further determine the caspase specificity of these probes, the CD2 or CD3 protein was incubated with either caspase-2 or caspase-3 for $1.5\mathrm{h}$ , and the products were analyzed by Western blot [Fig. 2c]. We observed that CD2 proteins were cleaved by caspase-2 after a $1.5\text{‐}\mathrm{h}$ incubation period [Fig. 2c, lane 2], while CD3 proteins were not cleaved by caspase-2 under the same conditions [Fig. 2c, lane 1]. Similarly, CD3 proteins can be cleaved by caspase-3 [Fig. 2c, lane 4] but cannot be cleaved by caspase-2 [Fig. 2c, lane 3]. These results indicate that the CD2 and CD3 are recognized uniquely by the activated caspase-2 and caspase-3, respectively.

Fig. 1

Schematic representation of the biosynthetic FRET probes. The linker of CD2 is 17-amino-acid peptide MHDQLTEE $\underset{̱}{\mathit{\text{VDVAD}}}$ GSEL, which contains a caspase-2 recognition site sequence. It is different from CD3 (a 16-amino-acid peptide MHDQLTEE $\underset{̱}{\mathit{\text{DEVD}}}$ GSEL with a linker containing a caspase-2 recognition site sequence). (The cleavage site is indicated by an underline.)

Fig. 2

Characterization of the FRET probes. (a) The emission spectra were measured using a spectrofluorometer (excitation $\text{wavelength}=433\mathrm{nm}$ ) while the recombinant CD2 protein was incubated with caspase-2 (final $\text{concentration}=2\text{unit}∕\mathrm{ml}$ ) for $1.5\mathrm{h}$ . (b) The emission spectra were measured using a spectrofluorometer (excitation $\text{wavelength}=433\mathrm{nm}$ ) while the recombinant CD3 protein was incubated with caspase-3 (final $\text{concentration}=2\text{unit}∕\mathrm{ml}$ ) for $1\mathrm{h}$ . (c) Western blot analysis of the recombinant CD2 and CD3 proteins exposed by activated caspases; the purified CD2 protein was incubated with caspase-3 (lane 1) and caspase-2 (lane 2) for $1.5\mathrm{h}$ , and the purified CD3 protein was incubated with caspase-2 (lane 3) and caspase-3 (lane 4) for $1.5\mathrm{h}$ (final $\text{concentration}=2\text{unit}∕\mathrm{ml}$ ).

3.2.

Caspase Assay for CD2 and CD3 in Apoptotic HeLa Cell Extract

Cisplatin is one of the most effective and widely used anticancer drugs. Recent studies have indicated that caspase-2 (Ref. 6) and caspase-3 (Ref. 24) were activated in cisplatin-treated HeLa cells. To determine the sensitivity of CD2 and CD3 to caspases during apoptosis, the two FRET probes were each transfected into their respective HeLa cells. HeLa cells that stably expressed the recombinant FRET probes were then treated with cisplatin $(10\mu \mathrm{g}∕\mathrm{ml})$ . Figures 3a and 3b display the emission spectra of cisplatin-treated and cisplatin-untreated HeLa cells, which were transfected with CD2 or CD3 and were excited at $433\mathrm{nm}$ . The cisplatin-untreated group peaked at $585\mathrm{nm}$ , but the cisplatin-treated groups did not peak at this level. To provide evidence that caspases cleaved the FRET probe in living HeLa cells, we applied Western blot analysis of cellular extracts after $7\text{to}12\mathrm{h}$ of cisplatin treatment, showing that the CD3 was efficiently cleaved into two monomers, CFP and DsRed [Fig. 3c, lane 2]. Similarly, the efficiency of cleavage could be detected in the cells transfected by CD2 [Fig. 3d, lane 2]. In the control group of cisplatin-untreated HeLa cells, however, no cleavage was detected [Fig. 3c, lane 1, and Fig. 3d, lane 1].

Fig. 3

Properties of CD2 and CD3 in cisplatin-induced apoptosis. (a) HeLa cells that stably expressed the CD2 fusion proteins were incubated with cisplatin $(10\mu \mathrm{g}∕\mathrm{ml})$ for $7\text{to}12\mathrm{h}$ . The cell suspensions were then subjected to spectral analysis with excitation wavelength at $433\mathrm{nm}$ . (b) HeLa cells that stably expressed the CD3 fusion proteins were incubated with cisplatin $(10\mu \mathrm{g}∕\mathrm{ml})$ for $7\text{to}12\mathrm{h}$ . The cell suspensions were then subjected to spectral analysis with excitation wavelength at $433\mathrm{nm}$ . (c) Cleavage assay of CD2 in lysates, prepared from cisplatin-treated HeLa cells which stably expressed CD2; its cleavage was then examined by Western blotting using an anti-GFP antibody. Lane 1, the lysates were prepared from cisplatin-treated HeLa cells. Lane 2, the lysates were prepared from cisplatin-untreated HeLa cells. Lane 3, the lysates were prepared from the CFP expressing HeLa cells. (d) Cleavage assay of CD3 in lysates, prepared from cisplatin-treated HeLa cells that stably expressed CD3; its cleavage was then examined by Western blotting using an anti-GFP antibody. Lane 1, the lysates were prepared from cisplatin-treated HeLa cells. Lane 2, the lysates were prepared from cisplatin-untreated HeLa cells.

3.3.

FRET Imaging of the Dynamics of Caspase-2 and Caspase-3 Activation in Living HeLa Cells during Cisplatin-Induced Apoptosis

To monitor caspase-2 or caspase-3 activation in living cells, HeLa cells that stably expressed the CD2 or CD3 fusion proteins were kept in a 96-well plate for $24\mathrm{h}$ , and imaging analysis was started immediately after the cells were exposed to cisplatin $(30\mu \mathrm{g}∕\mathrm{ml})$ . Using a three-filter imaging system, the time series of the ratio of FRET images of CD2 and CD3 expressing HeLa cells are shown in Figs. 4a and 5a , respectively. The FRET efficiency of CD2 and CD3 was significantly decreased in response to the cleavage of the probe. The values of $R_{\mathrm{FRET}}$ were obtained from the ROI in the different cells and were plotted as a function of time [Figs. 4b and 5b]. From these figures, we can observe that the activation of caspase-2 and caspase-3 was completed before the cell changed morphologically (such as membrane blebbing and cell shrinkage). Furthermore, once the activation of caspase-3 initiated in the cytosol, the activation process completed rapidly in $10\min$ [Fig. 5b]. However, in comparison to the dynamics of caspase-3 activation, caspase-2 activation occurs very slowly but ends earlier [Fig. 4b].

Fig. 4

Single-cell imaging FRET changes of CD2 in response to caspase activation during cisplatin-induced apoptosis in living HeLa cells. (a) Ratio of FRET images of the CD2 expressing HeLa cells. HeLa cells that stably expressed the CD2 fusion proteins were cultured in a 96-well plate for $24\mathrm{h}$ ; imaging analysis was started immediately after exposure to cisplatin $(30\mu \mathrm{g}∕\mathrm{ml})$ . Scale bar, $20\mu \mathrm{m}$ . (b) Dynamics of activation of caspase-2 in individual HeLa cells during cisplatin-induced apoptosis as examined in (a). The values of $R_{\mathrm{FRET}}$ are from the ROI, which is indicated by a circle.

Fig. 5

Single-cell imaging FRET changes of CD3 in response to caspase activation during cisplatin-induced apoptosis in living HeLa cells. (a) Ratio of FRET images of the CD3 expressing cells; HeLa cells that stably expressed the CD3 fusion proteins were cultured in a 96-well plate for $24\mathrm{h}$ ; imaging analysis was started $40\min$ after exposure to cisplatin $(30\mu \mathrm{g}∕\mathrm{ml})$ . Scale bar, $20\mu \mathrm{m}$ . (b) Dynamics of activation of caspase-3 in individual HeLa cells during cisplatin-induced apoptosis as examined in (a). The values of $R_{\mathrm{FRET}}$ are from the ROI, which is indicated by a circle.