Department of Biology, The Hong Kong University of Science and
Technology, Clear Water Bay, Hong Kong, China
*
Author for correspondence (e-mail:
bochang{at}ust.hk
)
Accepted May 2, 2001
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SUMMARY |
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Key words: Apoptosis, Cytochrome c, Mitochondria, GFP, Programmed cell death
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INTRODUCTION |
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This hypothesis has been widely cited and debated in the literature
(Desagher and Martinou, 2000;
Eskes et al., 1998
; Green and
Reed, 1998
; Heiskanen et al.,
1999
;
Jürgensmeier et al.,
1998
; Kluck et al.,
1997
; Kluck et al.,
1999
; Krohn et al.,
1999
; Matsuyama et al.,
2000
; Minamikawa et al.,
1999
; Narita et al.,
1998
; Pastorino et al.,
1998
; Pastorino et al.,
1999
; Scarletta et al.,
2000
; Shimizu et al.,
1999
; Vander Heiden et al.,
1997
; von Ahsen et al.,
2000
; Yang et al.,
1997
), but so far, there is
still a lack of conclusive evidence to prove or disprove it. First, the
supporting evidence for the mitochondrial swelling theory is based mainly on
indirect observations. For example, specific inhibitors of the PT pore such as
cyclosporin A or bongkrekic acid have been found to prevent apoptosis in
hepatocytes and other cell models (Bradham et al.,
1998
; Kroemer et al.,
1997
), while PT pore-opening
agent like attractyloside or Ca2+ have been observed to induce
matrix swelling and apoptosis (Jürgensmeier et
al., 1998
; Narita et al.,
1998
; Pastorino et al.,
1999
). Conflicting data,
however, have also been reported and suggest that cytochrome c release may not
be related to PT pore opening. For example, Eskes et al. observed that
Bax-induced cytochrome c release could not be inhibited by either cyclosporin
A or bongkrekic acid in isolated mitochondria (Eskes et al.,
1998
).
Second, there is a controversy about whether or not the mitochondria indeed
swell during apoptosis. Some studies have reported the observation of
mitochondrial swelling (Narita et al.,
1998; Vander Heiden et al.,
1997
), while others have
reported that swelling never occurred (Kluck et al.,
1999
; von Ahsen et al.,
2000
). Third, most previous
studies have been carried out using fixed cells or isolated mitochondria
(Antonsson et al., 2000
; Eskes
et al., 1998
;
Jürgensmeier et al.,
1998
; Kluck et al.,
1999
; Narita et al.,
1998
; Pastorino et al.,
1999
; Vander Heiden et al.,
1997
; von Ahsen et al.,
2000
), which could become
swelled during the fixation or isolation processes (Scheffler,
1999
). Fourth, even if
mitochondria are observed to swell during apoptosis, it is still not clear
whether the mitochondrial swelling is the cause or the result of cytochrome c
release.
In order to overcome these difficulties and directly test the mitochondrial swelling theory, one must study the events of cytochrome c release and mitochondrial swelling in an intact living cell. Thus, we have used digital imaging techniques to measure the dynamic re-distribution of GFP labeled cytochrome c during UV-induced apoptosis in living HeLa cells, and used a red color fluorescent dye, Mitotracker, to monitor the morphological change of mitochondria at the same time. The objective of our study is to answer two important questions: (1) do mitochondria swell during apoptosis?; and (2) if they do, is the mitochondrial swelling the cause or consequence of cytochrome c release?
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MATERIALS AND METHODS |
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Cell treatments and induction of apoptosis
HeLa cells were grown on glass coverslips at 37°C in a humidified
atmosphere containing 5% CO2 in modified Eagle's Medium (MEM)
supplemented with 10% fetal bovine serum (FBS) plus 100 µg/ml penicillin
and 100 U/ml streptomycin. To image cytochrome c, cells were transfected with
the cytochrome c-GFP plasmid using an electroporation method (Chang,
1997) and were allowed to
express the fusion gene for 48-60 hours. Apoptosis was induced either by
exposing cells to UV irradiation (300 µW/cm2) for 3 minutes, by
adding 10 ng/ml TNF
plus 10 µg/ml cycloheximide, or by adding 1
µM Actinomycin D to the medium. In the experiment using z-VAD-fmk (100 nM)
and cyclosporine A (5 µM), both chemicals were added 1 hour before UV
irradiation and kept in the medium throughout the experimental process.
Imaging methods
For living cell measurements, we used a laser scanning confocal microscope
(BioRad MRC-600) equipped with a krypton/argon laser to image cytochrome c-GFP
distribution and mitochondria morphology in cells grown on a coverslip. Laser
lines of 488 nm and 568 nm were used to observe cytochrome c-GFP and
Mitotracker, respectively. To stain mitochondria, Mitotracker Red CMXRos was
added to MEM to a final concentration of 0.5 µM and cells were incubated
for 5 minutes followed by washing with MEM once. After UV irradiation, the
coverslip was mounted onto a chamber containing an observation medium
(Hepes-buffered Dulbecco's MEM containing 4 mM glutathione, 1 mM L-ascorbic
acid, 0.5 mM DTT, pH 7.4). The cells were examined under the confocal
microscope with a heating box that maintained the temperature at 37°C.
For immunostaining studies, cells were exposed to the apoptosis-inducing
treatment first. After a waiting period, we washed cells and fixed them with
4% paraformaldehyde plus 0.1% glutaraldehyde for 20 minutes at room
temperature. Then, cells were immunostained using the standard method (Li et
al., 1999). The primary
antibody used was mouse anti-native cytochrome c (1:200 dilution), while the
secondary antibody was goat anti-mouse IgG conjugated with FITC (1:200
dilution). Mitochondria were stained with Mitotracker.
Quantitative analysis of cytochrome c release and morphological
change in mitochondria
We used the MetaMorph 3.0 software (Universal Image, West Chester, PA) to
analyze digital images recorded by the confocal microscope at different times
during apoptosis. For the mitochondrial diameter measurement, we drew a line
perpendicular to the mitochondrion in the magnified Mitotracker image and
determined its diameter based on the line-scan profile. Several line-scan
measurements were made on a single mitochondrion to obtain its average
diameter. This procedure was repeated on many mitochondria within the same
cell to obtain a statistical value. To quantify the distribution of cytochrome
c, we examined the pixel distribution profile of the cytoc-GFP image and
separated the mitochondrial signals from the cytosolic signals by choosing a
proper threshold (see Fig. 3).
Then, we integrated all pixels in the cell (Ftotal) and in the
mitochondria (Fmito). The ratio of mitochondrial pixels to
cytosolic pixels, Fmito/(Ftotal
Fmito), was used as a parameter to characterize the cytochrome
c-GFP distribution between mitochondria and the cytosol.
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RESULTS |
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Cytochrome c release occurs before mitochondrial swelling in
UV-induced apoptosis
In order to measure directly the event of cytochrome c release from
mitochondria during apoptosis, we used GFP-labeled cytochrome c to monitor the
dynamic re-distribution of cytochrome c in intact living cells. The GFP-tagged
cytochrome c protein (cytoc-GFP) was produced by expressing a plasmid vector
containing the cytochrome c-GFP fusion gene in HeLa cells (Mahajan et al.,
1998). Using a confocal
microscope operated under the two-channel measurement mode, we imaged both the
distribution pattern of cytoc-GFP and that of Mitotracker simultaneously. We
exposed the cells to UV light (300 µW/cm2) for 3 minutes to
induce apoptosis and then recorded the distribution of cytochrome c and
Mitotracker at different times. Fig.
2 shows a sample record of this time series measurement, where two
neighboring cells were caught to undergo cytochrome c release at slightly
different times. Before cytochrome c was released from mitochondria, the
distribution patterns of both cytochrome c and Mitotracker were the same as
that of mitochondria in the normal cell, i.e., they appeared as filamentous
structures (Fig. 2A). As
cytochrome c was released from mitochondria of the lower cell, the cytoc-GFP
in that cell became uniformly distributed over the entire cytoplasm
(Fig. 2B), while the
Mitotracker image shows that the morphology of mitochondria still remained
filamentous (Fig. 2B). Within
about 10 minutes of cytochrome c was release, mitochondria within the lower
cell were found to swell and their morphology became spherical
(Fig. 2C).
|
Similar results were observed in the upper cell in Fig. 2. Before cytochrome c was released from mitochondria, the distribution patterns of both cytoc-GFP and Mitotracker were the same and they appeared as filamentous structures (Fig. 2A-C). Cytochrome c release was found to be a highly synchronized event. At 171 minutes after the UV treatment, none of the mitochondria in the upper cell had released their cytochrome c (Fig. 2C). Five minutes later, however, cytochrome c was found to be released from about half of the mitochondria (Fig. 2D). Another minute later, all mitochondria had finished releasing their cytochrome c (Fig. 2E). It is clear from Fig. 2E (and Fig. 2B) that after cytochrome c was released from mitochondria, the great majority of mitochondria had not swelled. The swelling did occur later. For example, 13 minutes after cytochrome c release, mitochondria in the upper cell gradually changed from filamentous shapes to spherical shapes (Fig. 2F).
In order to demonstrate the morphological change of individual mitochondrion more clearly, we magnified the Mitotracker images within Fig. 2C,D,F and showed them in Fig. 2G-I, respectively. From these magnified images, it is clearly evident that mitochondria did not swell before or during cytochrome c release (Fig. 2G,H). Instead, mitochondria became swollen only after cytochrome c was released (Fig. 2I).
Quantitative analysis of the dynamics of cytochrome c release and
mitochondria swelling
To examine the temporal relationship between cytochrome c release and
mitochondrial swelling, we conducted a quantitative analysis of the
time-dependent change of the distribution of cytochrome c and the
morphological change of mitochondria within a single living cell. The method
is outlined in Fig. 3. First,
we determined the diameter of the mitochondria from the magnified Mitotracker
images using a line-scan method (Fig.
3A-C). The diameter of a mitochondrion was taken as the half width
of its pixel profile based on the line-scan measurement
(Fig. 3C). Several line-scan
measurements were made on a single mitochondrion to obtain its average
diameter. This procedure was repeated on many mitochondria within the same
cell to obtain a statistical value. Second, to quantify the distribution of
cytochrome c, we examined the pixel distribution profiles of the cytoc-GFP
image in two sample regions of the cell: Region 1 contained only cytoplasm,
while Region 2 contained both cytoplasm and mitochondria
(Fig. 3D). By comparing the
pixel distribution profiles of these two regions, we could choose a proper
threshold to separate the fluorescence signal contributed by cytoc-GFP located
in the mitochondria from that of the cytosolic cytoc-GFP (for more details,
see Materials and Methods).
We conducted this quantitative analysis for a stack of images recorded at different time on a single HeLa cell undergoing UV-induced apoptosis. The results are shown in Fig. 4A. The release of cytochrome c from mitochondria to cytoplasm was a very rapid and highly synchronized event. We found that mitochondria started to swell only after their cytochrome c had been released. This swelling process was less synchronized than the release of cytochrome c; some mitochondria swelled almost immediately after their cytochrome c was released, while others waited for several minutes before they started to swell. In a typical cell, it took roughly 14 minutes to convert all mitochondria within the cell from a filamentous shape to a spherical shape (Fig. 4A). This relatively poor synchrony also contributed to large error bars in the measurement of the mitochondrial diameter during this swelling period.
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To obtain a more detailed picture on the temporal relationship between cytochrome c release and mitochondrial swelling at a single-organelle level, we have further analyzed our images on individual mitochondria. In the periphery region of the cell where the concentration of mitochondria was less dense, one could measure both the cytochrome c distribution and morphological change in a single mitochondrion. For example, the mitochondrion marked by an arrowhead in Fig. 2D,E was shown to release its cytochrome c first without undergoing a morphological change. Results of a quantitative analysis of its cytochrome c release and change in diameter as a function of time are summarized in Fig. 4B. It is clear from this single-mitochondrion measurement that mitochondrial swelling occurred after cytochrome c was released.
Results of immunostaining studies also confirm that cytochrome c was
released before mitochondria swelled during apoptosis
In the above living cell study, the distribution of cytochrome c was
measured by imaging the fluorescence signal of the GFP-tagged cytochrome c.
There is a remote possibility that, for some unexpected reasons, the
re-distribution of endogenous cytochrome c from mitochondria might differ from
that of the GFP-tagged cytochrome c. To rule out this possibility, we have
conducted an immunostaining study. Here, HeLa cells (not transfected with the
cytochrome c-GFP fusion gene) were fixed at different times following an
UV-induced apoptotic treatment. The distribution of cytochrome c was
visualized by immunostaining the cells with anti-cytochrome c antibody. The
morphology of mitochondria was revealed by staining the cells with Mitotracker
Red. As we compared the distribution pattern of the endogenous cytochrome c
(as revealed by anti-cytochrome c) and the morphological pattern of
mitochondria, we found that the HeLa cells subject to the apoptotic treatment
could be classified into three different categories: (1) cells in which
cytochrome c was distributed mainly in the mitochondria, which were in the
normal filamentous shape (Fig.
5A); (2) cells in which cytochrome c was distributed in the
cytosol, and yet, their mitochondria remained in their filamentous shape and
did not swell (Fig. 5B); and
(3) cells in which cytochrome c was distributed in the cytosol, while their
mitochondria were in the swollen spherical shape
(Fig. 5C). Category 1 cells are
believed to be those that their cytochrome c had not yet been released from
mitochondria to cytosol. Category 3 cells are those that their cytochrome c
was released some time ago and their mitochondria had become swollen. The
existence of the Category 2 cells is most interesting; it shows that when the
endogenous cytochrome c was released from mitochondria to the cytosol, their
mitochondria had not yet started to swell. This result confirms the finding of
our living cell measurements that cytochrome c release occurred before
mitochondrial swelling.
|
The statistical distribution of these three categories of cells is shown in Table 1. For control cells that were not treated with UV irradiation, 99.2% of cells were found to belong to Category 1. Only 0.8% of cells were in Category 3, which represented a very small population of dead cells normally found in the cell culture. No cell in Category 2 was observed. The distribution was quite different in UV-treated cells. Three hours after UV treatment, a large number of cells were found to belong to Category 3, and the population of Category 2 cells was significantly larger than zero. The fact that the population of these Category 2 cells was much smaller than those of the other two categories indicates that the time window between the events of cytochrome c release and mitochondrial swelling must be relatively short. This finding, again, is consistent with the results of our living cell measurement.
|
To test whether our finding was unique in UV-induced apoptosis or a general phenomenon, we repeated the immunostaining study using various apoptotic inducing treatments, including TNF (Fig. 5D), actinomycin D (Fig. 5E) and staurosporin (data not shown). Their results were all consistent with what we found in UV-induced apoptosis. Regardless of the type of apoptotic treatment, when we examined the morphology of mitochondria and the distribution pattern of cytochrome c in a population of fixed HeLa cells that were undergoing apoptosis, we always found three categories of cells similar to those observed in UV-induced apoptosis (Table 1). In other words, there was always a small percentage of fixed cells in which cytochrome c was distributed in the cytoplasm and the mitochondria were in filamentous shape. Their existence implies that cytochrome c release must occur before mitochondrial swelling during apoptosis.
z-VAD-fmk and cyclosporin A could not inhibit cytochrome c release or
mitochondrial swelling
We had applied caspase inhibitor z-VAD-fmk to UV-treated cells and found
that z-VAD-fmk could block apoptosis. The application of z-VAD-fmk, however,
could not inhibit cytochrome c release
(Table 1). This observation is
in agreement with that reported earlier by Goldstein et al. (Goldstein et al.,
2000). In our experiment, we
found that z-VAD-fmk could not block mitochondria swelling either. Application
of a PT pore inhibitor, cyclosporin A, also failed to block UV-induced
apoptosis, cytochrome c release or mitochondria swelling
(Table 1). This result further
argued against the hypothesis that opening of the PT pore (to cause matrix
swelling) is required for cytochrome c release.
Treatment of CCCP indicated that mitochondria swelling alone could
not induce cytochrome c release
To demonstrate that mitochondrial swelling alone cannot cause cytochrome c
to release, we have conducted another living cell imaging experiment. After we
expressed the GFP-cytochrome c fusion gene in HeLa cells and labeled them with
Mitotracker (Fig. 6A), we
applied carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) to induce
mitochondrial swelling. Treatment of CCCP is known to cause a short circuit
effect in the proton gradient of the mitochondrial inner membrane and induce
mitochondria to swell (Minamikawa et al.,
1999). Before application of
CCCP, mitochondria were in the normal filamentous shape
(Fig. 6A). Eight minutes after
CCCP application, some mitochondria were seen to start to swell
(Fig. 6B). After 19 minutes,
most mitochondria had become swollen and changed into spherical shapes
yet cytochrome c was not released (Fig.
6C). In fact, cytochrome c was found to remain in the mitochondria
even after mitochondria had been in the swollen state for more than 1 hour
(Fig. 6D). Furthermore, this
CCCP-induced mitochondrial swelling was reversible. Mitochondria returned to
their filamentous structure after CCCP was removed and the cell was found to
recover eventually. This result shows that mitochondrial swelling alone cannot
cause cytochrome c to release or induce apoptosis.
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DISCUSSION |
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The degree of mitochondrial swelling after cytochrome c release appeared to be relatively mild and did not necessarily cause a rupture of its outer membrane. From the simple mathematical model shown in Fig. 1C, we estimated that when a mitochondrion swells by changing to a spherical shape, it can increase its diameter by 2.34-fold without increasing its surface area. In the experiment shown in Fig. 2, the average diameter of mitochondria was found to increase to 2.21-fold after cytochrome c release. The extent of mitochondria swelling varied slightly from cell to cell; its average was 1.99±0.17 over six cells that we analyzed. This observed diameter change was within the theoretical limit of the non-stretching model (Fig. 1C). Thus, our data suggest that, even though mitochondria did swell after cytochrome c release, the swelling probably did not cause their outer membrane to rupture. Alternatively, this relatively small change in diameter may indicate that, after the release of cytochrome c, the filamentous mitochondria could be broken into shorter pieces before swelling.
If mitochondrial swelling was not the cause of cytochrome c release, then
what would facilitate cytochrome c to redistribute during apoptosis? There are
several alternative models available at present (Desagher and Martinou,
2000). Cytochrome c could be
released through specific channels, such as Bax channel or Bax-VDAC channel,
or through lipid pore (or protein-lipid complex) at the outer membrane of
mitochondria (Martinou et al.,
2000
). Some recent studies
have presented evidence to support these channel models (Antonsson et al.,
2000
; Basanez et al.,
1999
; Shimizu et al.,
1999
). For example, Shimizu et
al. have reported that cytochrome c can pass through VDAC channel incorporated
into lipid vesicles with the help of Bax (Shimizu et al.,
1999
). There is still a
problem, however, with this model. The VDAC channel has a relative small
opening; proteins like GST-GFP (50 kDa) could not pass through the VDAC
channel (Shimizu et al.,
1999
). But during apoptosis,
many proteins in the intermembrane space, including AIF (57 kDa), were
released (Kluck et al., 1999
;
Susin et al., 1999
). At this
point, we feel that protein-lipid complex formed by Bax (or other Bcl-2 family
proteins) insertion into the outer mitochondrial membrane may offer the most
attractive model.
Another question is what caused mitochondria to swell after cytochrome c
was released. Such mitochondrial swelling was probably not caused by a caspase
feedback loop, as we found that caspase inhibitor z-VAD-fmk could not block
mitochondria swelling (Table
1). As the PT pore inhibitor CsA also failed to block the
swelling, one may suggest that reactive oxygen species (ROS) generated after
cytochrome c release might play specific roles. It has been reported earlier
that ROS can oxidize lipids and proteins in the inner membrane of mitochondria
to cause membrane permeabilization and mitochondria swelling (Kowaltowski et
al., 1996).
In conclusion, we have shown that during apoptosis, release of cytochrome c
occurred before mitochondria swelling. The interval between the two events was
relatively short (within about 10 minutes). Thus, release of cytochrome c
could not be caused by rupturing the outer mitochondrial membrane due to
matrix swelling. This conclusion holds true for apoptosis induced by various
treatments including UV, TNF, actinomycin D and staurosporin. It is possible
that, the mitochondrial swelling model might play a more appropriate role in
the process of necrosis (Trump,
1981).
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ACKNOWLEDGMENTS |
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