Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 37-45
doi:10.1111/j.1745-7270.2007.00246.x
Fluorescence Resonance Energy
Transfer Analysis of Bid Activation in Living Cells during Ultraviolet-induced
Apoptosis
Yinyuan WU, Da XING*, Lei LIU,
Tongsheng CHEN, and Wei R. Chen
MOE Key Laboratory of Laser Life
Science & Institute of Laser Life Science, South China Normal University,
Guangzhou 510631, China
Received: August 23, 2006
Accepted: November 10, 2006
This work was supported by the grants from the National Natural Science
Foundation of China (No. 60378043 and No. 30470494), and the Natural Science
Foundation of Guangdong Province (No. 015012, No. 04010394 and No.
2004B10401011)
*Corresponding
author: Tel, 86-20-85210089; Fax, 86-20-85216052; E-mail, [email protected]
Abstract Ultraviolet (UV) irradiation is a
DNA-damage agent that triggers apoptosis through both the membrane death
receptor and mitochondrial apoptotic signaling pathways. Bid, a pro-apoptotic
Bcl-2 family member, is important for most cell types to apoptose in response
to DNA damage. In this study, a recombinant plasmid, YFP-Bid-CFP, which was
comprised of yellow and cyan fluorescent protein and a full length Bid, was used
as a fluorescence resonance energy transfer analysis (FRET) probe. Using the
FRET technique based on YFP-Bid-CFP, we found that Bid activation was initiated
at 91 h
after UV irradiation, and the average duration of the activation was 7510 min. Bid
activation coincided with a collapse of the mitochondrial membrane potential
with an average duration of 5010 min. When
cells were pretreated with Z-IETD-fmk (caspase-8 specific inhibitor) the
process of Bid activation was completely inhibited, but the apoptosis was only
partially affected. Z-DEVD-fmk (caspase-3 inhibitor) and Z-FA-fmk (non asp
specific inhibitor) did not block Bid activation. Furthermore, the endogenous
Bid activation with or without Z-IETD-fmk in response to UV irradiation was
confirmed by Western blotting. In summary, using the FRET technique, we
observed the dynamics of Bid activation during UV-induced apoptosis and found
that it was a caspase-8 dependent event.
Key words ultraviolet irradiation; Bid activation;
apoptosis; caspase-8; fluorescence resonance energy transfer analysis
Bid was first reported in 1996 and is widely expressed in various
tissues, with the highest level in the kidney [1]. In a resting cell, Bid is
predominantly cytoplasmic. Following tumor necrosis factor-a (TNF-a) or Fas
treatment, Bid is cleaved by caspase-8 in an unstructured loop, exposing a new
amino terminal glycine residue, which becomes myristoylated, facilitating its
translocation to the mitochondria where it induces the activation of Bax and
Bak, resulting in the release of cytochrome c [2,3]. However,
the apoptotic pathways in which Bid plays a role are not yet fully
characterized. Studies with Bid–/– mice have
demonstrated that Bid is required for Fas-induced apoptosis [4]. On the other hand,
Bid–/– mouse
embryonic fibroblasts (MEFs) were found to be as susceptible as Bid+/+ MEFs to a wide range of intrinsic damage signals [5]. Recently, it
was demonstrated that Bid–/– MEFs are less susceptible than Bid+/+ MEFs
to adriamycin, a DNA-damage reagent, as well as to the nucleotide analog
5-fluorouracil [6]. Thus, Bid might contribute to the DNA-damage response.Ultraviolet (UV) irradiation has multiple cellular targets that
trigger different signaling cascades leading to apoptosis. UV irradiation is a DNA-damage agent that activates a p53-dependent apoptotic response [7,8]. DNA damage can change the phosphorylation levels of
p53 protein resulting in cell cycle arrest and apoptosis. p53
stimulates a wide network of signals that act through two major apoptotic
pathways. The extrinsic pathway is initiated through ligation of the death
receptor family receptors by their respective ligands. This family includes
the tumor necrosis factor receptors, CD95/Fas/APO-1 and the TRAIL receptors
[9,10]. Receptor ligation is followed by the formation of the death-inducing
signaling complex (DISC), which is composed of the adapter molecule FADD and
caspase-8 [11,12]. Recruitment to DISC activates caspase-8, which in turn
either directly cleaves and activates the effector caspases, or indirectly
activates the downstream caspases through cleavage of the BH3 protein Bid,
leading to engagement of the intrinsic pathway of apoptosis [13–16]. The
intrinsic pathway of caspase activation is regulated by the pro- and anti-apoptotic
Bcl-2 family proteins. These proteins induce or prevent the release of
apoptogenic factors, such as cytochrome c and Smac/DIABLO, from the
mitochondrial intermembrane space into the cytosol [17–20].Fluorescence resonance energy transfer (FRET) is a process by which
transfer of energy occurs from a donor fluorophore molecule to an acceptor
fluorophore molecule in close proximity. The emission spectrum of the donor
molecule overlaps with the absorption spectrum of the acceptor molecule. When the
two fluorophores are spatially close enough, there is energy transfer
between the donor and acceptor molecules. The excited donor transfers its
energy to the acceptor, which results in a reduction in donor fluorescence
emission and, at the same time, an increase in acceptor fluorescence emission
[21]. Thus, FRET is a powerful technique that can
provide insight into the spatial and temporal dynamics of protein-protein
interactions in vivo [22–26]. Recently, a fusion protein YFP-Bid-CFP, which was constructed
by connecting cyan fluorescent protein (CFP) and yellow fluorescent protein
(YFP) to the C terminus and N terminus of Bid, respectively, has been used to
observe the dynamics of Bid activation [27].In this study, to investigate Bid activation induced by UV
irradiation, we transfected ASTC-a-1 cells with YFP-Bid-CFP and examined the
dynamics of Bid activation at the single cell level, which was confirmed by
fluorescence spectroscopy and Western blotting analysis. Our findings extend
the knowledge about the cellular signaling mechanisms mediating UV-induced
apoptosis.
Materials and Methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco
(Grand Island, USA). Z-IETD-fmk (caspase-8 inhibitor), Z-DEVD-fmk (caspase-3 inhibitor)
and Z-FA-fmk (non asp specific inhibitor) were purchased from BioVision
(Mountain View, USA). Lipofectamine reagent, recombinant human TNF-a and
cycloheximide (CHX) were purchased from Invitrogen (Carlsbad, USA). DNA
extraction kit was purchased from Qiagen (Valencia, USA). YFP-Bid-CFP was
kindly supplied by Dr. K. TAIRA [27]. Other chemicals were mainly from Sigma (St. Louis, USA).
Cell culture and treatment
The human lung adenocarcinoma cell line (ASTC-a-1) was obtained from
the Department of Medicine, Jinan University (Guangzhou, China) and cultured in
DMEM supplemented with 15% fetal calf serum (FCS), penicillin (100 U/ml) and
streptomycin (100 mg/ml) with 5% CO2 at 37 ?C in a humidified
incubator. Transfection was performed with Lipofectamine reagent according to
the manufacturer’s protocol. The medium was replaced with fresh culture medium
after 5 h. Cells were examined 24–48 h after transfection. For the generation of
stable cell lines, transfected cells were selected in the presence of G418 (1
mg/ml) for 2 weeks and fluorescent clones were enriched. For 120 mJ/cm2 UV irradiation, medium was removed, and cells were rinsed with
phosphate-buffered saline (PBS) and irradiated, and then medium was restored.
Cells were pretreated with Z-IETD-fmk (10 mM), Z-DEVD-fmk (40 mM) and Z-FA-fmk
(40 mM) for 1 h respectively before UV irradiation.
The inhibitors were kept in the medium throughout the experimental process.
FRET analysis
For analysis of Bid activation, cells were transfected with YFP-Bid-CFP
[27], and the dynamics of Bid activation was detected at the single cell level
by FRET analysis. FRET was performed on a commercial laser scanning microscope
(LSM510/ConfoCor2) combination system (Zeiss, Jena, Germany). For excitation,
the 458 nm line of an Ar-ion laser was attenuated with an acousto-optical
tunable filter, reflected by a dichroic mirror (main beam splitter HFT458),
and focused through a Zeiss Plan-Neofluar 40?/1.3 NA oil DIC objective onto the sample. CFP and YFP (FRET-acceptor)
emission was collected through 470–500 nm and 535–595 nm barrier filters,
respectively. The quantitative analysis of the fluorescence images was
performed using Zeiss Rel3.2 image processing software (Zeiss). After
background subtraction, the average fluorescence intensity per pixel was
calculated. The onset of Bid activation was defined as the time point at which
the YFP/CFP emission ratio irreversibly declined. During control experiments,
bleaching of the probe was negligible.For analysis of Bid activation, cells were transfected with YFP-Bid-CFP
[27], and the dynamics of Bid activation was detected at the single cell level
by FRET analysis. FRET was performed on a commercial laser scanning microscope
(LSM510/ConfoCor2) combination system (Zeiss, Jena, Germany). For excitation,
the 458 nm line of an Ar-ion laser was attenuated with an acousto-optical
tunable filter, reflected by a dichroic mirror (main beam splitter HFT458),
and focused through a Zeiss Plan-Neofluar 40?/1.3 NA oil DIC objective onto the sample. CFP and YFP (FRET-acceptor)
emission was collected through 470–500 nm and 535–595 nm barrier filters,
respectively. The quantitative analysis of the fluorescence images was
performed using Zeiss Rel3.2 image processing software (Zeiss). After
background subtraction, the average fluorescence intensity per pixel was
calculated. The onset of Bid activation was defined as the time point at which
the YFP/CFP emission ratio irreversibly declined. During control experiments,
bleaching of the probe was negligible.
Performance of acceptor
photobleaching
ASTC-a-1 cells transfected with YFP-Bid-CFP were grown on the
coverslip of a chamber. The chamber was placed on the stage of the LSM
microscope for performance of acceptor photobleaching. The acceptor photobleaching
was performed with the highest intensity of 514 nm laser, and the images of YFP
and CFP were recorded and processed with Zeiss Rel3.2 image processing
software.
Spectrofluorometric analysis
of Bid activation induced by UV irradiation in living cells
ASTC-a-1 cells stably expressing YFP-Bid-CFP were grown in DMEM
supplemented with 15% FCS for 48 h. Then the cells were treated with UV
irradiation at fluence of 120 mJ/cm2. After 12 h, the cells were
immediately transferred into a quartz cuvette, which was then placed inside the
sample holder of an LSM510 luminescence spectrometer (PerkinElmer, Boston,
USA). The fluorescence emission spectra were obtained by carrying out a
spectrum scanning analysis of the luminescence spectrometer. The excitation
wavelength was 434±5 nm, the excitation slit was 10 nm, the emission slit was
15 nm and the scanning speed was 200 nm/s. The corresponding background
spectra of cell-free culture medium were subtracted.
SDS-PAGE and Western blotting
analysis
Twelve hours after UV irradiation, cells were scraped from the dish,
washed twice with ice-cold PBS (pH 7.4), and lysed with ice-cold lysis buffer
[50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 100 mg/ml
phenylmethylsulphonyl fluoride (PMSF)] for 30 min on ice. The lysates were
centrifuged at 13,400 g for 5 min at 4 ?C,
and the protein concentration was determined. Equivalent samples (30 mg protein
extract) underwent 12% SDS-PAGE. The proteins were then transferred onto nitrocellulose
membranes, and probed with Bid monoclonal antibody (Cell Signaling Technology, Beverly, USA)
followed by rabbit primary antibodies conjugated to horseradish peroxidase
(KPL, Gaithersburg, USA).
Confirmation of cell apoptosis
ASTC-a-1 cells were cultured in a 96-well microplate at a density of
5?103 cells/well for 24 h. The cells were
then divided into five groups and exposed to UV irradiation at fluences of 0
(control), 30, 60, 120 and 240 mJ/cm2 for 12 h respectively.
Cell cytotoxicity was assessed with CCK-8 (Dojindo Laboratories, Kumamoto,
Japan) according to the manufacturer’s instructions. A450, the absorbance value at 450 nm, was read with a 96-well plate
reader (DG5032; Huadong, Nanjing, China), and the A450 was inversely proportional to the degree of cell apoptosis.To assess the changes in nuclear morphology typical of apoptosis,
ASTC-a-1 cells were cultured on 35 mm glass-bottomed dishes. After 12 h UV
irradiation, the cells were washed twice with PBS (pH 7.4). Subsequently, the
cells were stained with 1 mM Hoechst 33258 for 10 min at room temperature. The
cells were then washed twice with PBS and viewed under a Nikon fluorescent
microscope with a 330–380 nm band pass excitation filter and a 450–490 nm band pass emission
filter.
Statistical analysis
Data are represented as mean±SEM. Statistical analysis was carried
out with Student? paired t-test. Differences were considered statistically
significant at P<0.05.
Results
Characterization of
YFP-Bid-CFP in living ASTC-a-1 cells
Bid activation was monitored by the FRET technique using a fusion
protein YFP-Bid-CFP, which was comprised of YFP, CFP and the FL-Bid protein as
a linker.Before Bid was activated, CFP and YFP were covalently linked
together. Energy could be transferred directly from CFP to YFP, so fluorescence
emitted from YFP could be detected when CFP was excited. Once Bid was
activated, CFP was separated from YFP, so the FRET effect of YFP-Bid-CFP must
have decreased effectively [Fig. 1(A)]. Acceptor bleaching experiments
were carried out to assess the sensitivity of the FRET probe. Acceptor
photobleaching, one of the techniques for measuring FRET, the acceptor
molecule of the FRET pair was bleached, resulting in an unquenching of the
donor fluorescence [28]. The acceptor fluorophore YFP was selectively bleached
by repeated scanning of the cell area [Fig. 1(B)]. A quantitative
analysis of acceptor bleaching showed the absolute fluorescence intensities
for CFP and YFP for a single cell when plotted as a function of time [Fig.
1(B)]. On bleaching, there was a marked decrease in the acceptor
fluorescence (YFP), which coincided with an increase in the donor fluorescence
(CFP) because of an inability of the acceptor to accept energy from the donor
after bleaching. Out of the bleaching area, the intensities of CFP and YFP
remained unchanged (data not shown). Therefore, the increase of CFP fluorescence
by bleaching YFP confirmed that FRET exists between the two fluorescent
proteins in the YFP-Bid-CFP in vivo.
Real-time monitoring of Bid
activation during TNF-a-induced apoptosis in living
ASTC-a-1 cells
To define our system, we investigated Bid activation during TNF-a-induced
apoptosis. Cells were treated with 200 ng/ml TNF-a and 1 mg/ml CHX at the
start of the measurement. After 4 h and 55 min, the fluorescence intensity of
CFP increased and that of YFP decreased, which implied that YFP-Bid-CFP was
cleaved. The typical time-course images of YFP-Bid-CFP are shown in Fig.
2(A). The same FRET was confirmed by quantitative analysis of fluorescence
intensities [Fig. 2(B)].
Real-time monitoring of Bid
activation during UV-induced apoptosis in living ASTC-a-1 cells
To directly observe the activation of Bid during UV-induced
apoptosis, we transfected ASTC-a-1 cells with YFP-Bid-CFP, and then examined
the dynamics of Bid activation induced by UV irradiation at the single cell
level. The fluorescence intensity of CFP increased and YFP decreased at 9 h
after UV irradiation, which implied that YFP-Bid-CFP was cleaved. The typical
time-course images of YFP-Bid-CFP are shown in Fig. 3(A). The imaging
analysis commenced 8 h after UV irradiation, and the results showed that Bid
activation was initiated at 9 h after UV irradiation and reached its maximum
activation in 1 h [Fig. 3(B)]. To define the average initiation and
duration of Bid activation, we compared the dynamics of Bid activation in four
individual cells during UV-induced apoptosis. On average, the initiation of Bid
activation was 91 h after UV irradiation, and the duration of Bid activation
was 7510 min [Fig. 3(C)]. To determine whether Bid activation induced by UV irradiation was a
caspase-8 dependent event, cells were pretreated with various inhibitors for 1
h before UV
irradiation. In the presence of Z-IETD-fmk, the fluorescence
intensities of YFP, CFP and the ratio YFP/CFP remained unchanged, which indicated
that Bid activation was blocked by inhibiting caspases-8 activation [Fig.
3(D)]. In the samples treated with Z-DEVD-fmk and Z-FA-fmk, the results
were the same as that of UV treated cells respectively, which indicated that
Z-DEVD-fmk and Z-FA-fmk did not block Bid activation [Fig. 3(E)]. These
results revealed that the Bid activation was a caspase-8 dependent event.
Real-time detection of a
collapse of the mitochondrial membrane potential induced by UV irradiation in
living cells
To determine whether Bid activation coincided with a collapse of
mitochondrial transmembrane potential induced by UV irradiation, we
monitored a collapse of mitochondrial membrane potential using Rhodamine 123.
The typical time-course images of Rhodamine 123 are shown in Fig. 4(A).
The same results were confirmed by quantitative analysis of fluorescence
intensities in three individual cells [Fig. 4(B)]. On average, the
initiation of a collapse of mitochondrial transmembrane potential was 54030 min
after UV irradiation, and the duration of a collapse of mitochondrial
transmembrane potential was 5010 min.
Spectrofluorometric and
Western blotting analysis of Bid activation induced by UV irradiation in living
cells
To further demonstrate Bid activation induced by UV
irradiation, we used a spectrometer to measure the changes of FRET effects of
YFP-Bid-CFP in response to different treatments as indicated. The results of
spectrofluorometric analysis of the activation of YFP-Bid-CFP in living cells
are shown in Fig. 5(A). UV irradiation resulted in a decrease in FRET,
which indicated that Bid was activated, thus the emission peak of CFP (476 nm)
increased, and the emission peak of YFP (527 nm) decreased; and we did not
detect such results with UV irradiation in the presence of Z-IETD-fmk. These
results further confirmed that Bid activation was a caspase-8 dependent event,
which were consistent with the results from single cell imaging analysis. To find whether the endogenous Bid activation was the same as overexpression,
we used Western blotting analysis to study the endogenous Bid activation
during UV-induced apoptosis with or without Z-IETD-fmk treatment [Fig. 5(B)].
In the experiment, we used actin as a loading control. In the absence of
Z-IETD-fmk, cleaved Bid was detected 12 h after UV irradiation, in the presence
of Z-IETD-fmk, cleaved Bid was not detected 12 h after UV irradiation. Thus, it
suggested that Bid activation by UV irradiation was a caspase-8 dependent
event. The experiments were repeated three times.
UV irradiation induces
apoptosis in ASTC-a-1 cells
To establish a proper UV irradiation dose to induce apoptosis,
ASTC-a-1 cells were irradiated with various kinds of fluence. Apoptosis was
analyzed using a Cell counting kit-8 at 12 h after UV irradiation. The A450 value, an indicator of cell apoptosis, was measured. As shown in Fig.
6(A), the A450 value decreased as the
irradiation fluence increased. This indicated that UV irradiation caused a
dose-dependent increase in the percentage of apoptotic cells. A dose of 120
mJ/cm2 can induce a substantial number of cells at 12 h after UV
irradiation, and at the higher dose of 240 mJ/cm2, the
percentage of apoptotic cells decreases slightly.
To determine whether Bid activation is required for UV-induced
apoptosis or simply activated as a consequence of apoptosis, cell apoptosis was
analyzed using a Cell counting kit-8 at 12 h after 120 mJ/cm2 UV irradiation in the presence or absence of
Z-IETD-fmk, respectively. Fig. 6(B) indicates that Bid did not
contribute to UV-induced apoptosis.
To further confirm UV-induced apoptosis, we used Hoechst 33258
staining to observe cells at 12 h after 120 mJ/cm2 UV
irradiation. As shown in Fig. 6(C), cells exhibited typical apoptotic
nuclei at 12 h after UV irradiation judged by chromatin
condensation and nuclear fragmentation.
Discussion
UV irradiation is known to induce a cell death cascade involving
mitochondria, which eventually leads to apoptosis. However,
the precise initiating apoptotic mechanisms upstream of
mitochondria remain obscure. Bid activation can be conducted by several
proteases. Caspase-8 has been shown to be the major protease responsible for
Bid activation during death receptor-mediated apoptosis [13,15], and calpain is
also shown to cleave Bid [29,30]. Other reports demonstrate that Bid also can
be cleaved by caspase-3 and caspase-2 in the intrinsic pathway, which is
independent of death receptors [31–33]. To determine which protease contributes
to Bid activation during UV-induced apoptosis, we investigated Bid activation
in the presence of Z-IETD-fmk, Z-DEVD-fmk and Z-FA-fmk after UV irradiation. In
this study, we monitored for the first time the dynamics of Bid activation
during UV-induced apoptosis in living cells. Our results show that the effects
of UV irradiation on the ASTC-a-1 cells apoptosis depend on the dose of UV
irradiation [Fig. 6(A)]. We also show that Z-IETD-fmk did not block
UV-induced apoptosis [Fig. 6(B)]. When the cells were treated with UV
irradiation (120 mJ/cm2), a typical dosage of UV
irradiation to induce apoptosis of ASTC-a-1 cells in our conditions (Fig. 6),
both FRET imaging and spectrofluorometric analysis showed an increase in the
CFP emission and a corresponding decrease in the YFP emission. However, in the
presence of Z-IETD-fmk, there was no change in the CFP and YFP emission (Figs.
3 and 5). To further confirm these points, we used Western blotting
analysis to study the endogenous Bid activation during UV-induced apoptosis
with or without Z-IETD-fmk treatment [Fig. 5(B)], and the results were
consistent with FRET imaging and spectrofluorometric analysis. In addition, we
investigated collapse of mitochondrial transmembrane potential induced
by UV irradiation, which coincided with Bid activation (Fig. 4).FRET, a noninvasive technique, can spatio-temporally monitor
cellular events in different physiological conditions at a single cell level
[34–36].
It has been used to study enzyme activity, protein location, protein
translocation, small ligand binding, protein-protein interaction,
conformational change, and real-time posttranslational modification [22].
Specifically, FRET has been used to detect apoptotic signals that involve
activation of different caspases [37–39], interactions between Bcl-2 and Bax
[35,40], Ca2+ levels [36,41,42] and other protein activities. This can not be
fully elucidated by traditional biophysical or biochemical approaches, which
can only measure the average behavior of cell populations and the static
spatial information available from fixed cells and thus, can not provide direct
access to cells life events in their natural environment [22]. In our current study, we employed single-cell FRET analysis to
monitor the dynamics of Bid activation by UV irradiation. To our best
knowledge, this was the first time that the temporal and spatial profiles of
UV-induced apoptosis have been observed by FRET using YFP-Bid-CFP at the single
cell level. Our results demonstrated that Bid activation was initiated 91 h
after UV irradiation, and the average duration of the activation was 75±10 min, which
coincided with a collapse of the mitochondrial membrane potential, and Bid
activation was a caspase-8 dependent event during UV-induced apoptosis.
Acknowledgement
We thank
Dr. Taira (University of Tokyo,
Tokyo, Japan) for kindly providing the YFP-Bid-CFP plasmid.
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