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J Biol Chem, Vol. 274, Issue 9, 5654-5658, February 26, 1999
§,
,**
From the Departments of Anatomy, ¶ Physiology
and Biophysics, and Biomedical Engineering, School of Medicine,
Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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 Top
 Abstract
 Introduction
References
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Cytochrome c is released from
mitochondria into the cytosol in cells undergoing apoptosis. The
temporal relationship between cytochrome c release and loss
of mitochondrial membrane potential was monitored by laser-scanning
confocal microscopy in single living pheochromocytoma-6 cells
undergoing apoptosis induced by staurosporine. Mitochondrial membrane
potential monitored by tetramethylrhodamine methyl ester decreased
abruptly in individual cells from 2 to 7 h after treatment with
staurosporine. Depolarization was accompanied by cytochrome
c release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The
results show that mitochondrial depolarization accompanies cytochrome c release in pheochromocytoma-6 cells undergoing apoptosis.
Substantial evidence implicates mitochondria in apoptotic cell
death (1-4). It is thought that proteins normally restricted to the
mitochondrial intermembrane space, including cytochrome c
and the 50-kDa apoptosis-inducing factor, are released to the cytosol
where they initiate the apoptotic cascade (5-8). The mechansim of
protein release has not been established. One proposal is that protein
release requires rupture of the outer mitochondrial membrane and that
this is a consequence of the onset of the mitochondrial permeability
transition (MPT)1 (9, 10).
Onset of the MPT, which is an inner membrane process, would depolarize
the inner membrane (11). Thus, the MPT hypothesis implies a direct
temporal relationship between mitochondrial depolarization and
cytochrome c release. Experimental data addressing this
connection are limited.
Actually, two recent studies showed that cytochrome c
release during apoptosis was not accompanied by mitochondrial
depolarization (12, 13). In these studies, mitochondrial membrane
potential ( To examine the temporal relationship between cytochrome c
release and mitochondrial depolarization, we set out to monitor these
events in the individual mitochondria of single cells undergoing apoptosis. To monitor mitochondrial Plasmid Preparation--
Full-length cDNA sequence of rat
cytochrome c was synthesized by polymerase chain reaction
amplification using Pfu polymerase (Stratagene, La Jolla,
CA), and cloned into NheI and XhoI sites of the
pEGFP-N1 plasmid (CLONTECH Laboratories, Inc., Palo
Alto, CA). Polymerase chain reaction was performed using
oligonucleotide primers designed to introduce the KOZAK sequence and
remove the stop codon of cytochrome c. The KOZAK sequence of
the GFP in pEGFP-N1 was removed to ensure expression of only the
cytochrome c-GFP fusion protein.
Cell Line, Culture Conditions, and Transfections--
Rat
pheochromocytoma-6 (PC6) cells were cultured in Dulbecco's modified
Eagle's medium/F12 medium supplemented with 15 mM HEPES, 4 mM glutamine, 17.5 mM glucose, heat-inactivated
10% fetal bovine serum, 5% horse serum, and penicillin/streptomycin.
For transfection, cells (4 × 105) were plated on
poly-D-lysine-coated 40-mm glass coverslips. One day later,
cells were transiently transfected using 4 µg of plasmid DNA
(cytochrome c-GFP or GFP) and 24 µl of cationic
lipid Lipofect- AMINETM (Life Technologies, Inc.)
in 2 ml of serum free Opti-MEM medium (Life Technologies, Inc.) per
coverslip. After 3 h, an equal volume of regular medium
containing 2× normal serum was added. After 1 day, the cells were
placed in normal growth medium without antibiotics.
Western Blot Analysis--
Immunoblots were performed as
described before (21, 22). PC6 cells were harvested, washed twice with
ice-cold phosphate-buffered saline, and lysed with ice-cold lysis
buffer (150 mM NaCl, 1 mM EGTA, 1% Triton
X-100, 1% sodium deoxycholate, 50 mM Tris-HCl, pH 8.0)
containing protease inhibitors (0.2 µg/ml pepstatin, 0.2 µg/ml
leupeptin, 1 µg/ml aprotinin, and 0.2 mM
phenylmethylsulfonyl fluoride). The cell lysates were centrifuged at
10,000 × g for 10 min at 4 °C. Supernatants were
mixed with Laemmli sample buffer. Forty µg of protein each of
nontransfected cells and cytochrome c-GFP transfected cells,
and 1 µg of protein of GFP were separated on a 12%
SDS-polyacrylamide gel electrophoresis gel. The proteins were
transferred to a polyvinylidene difluoride membrane (Bio-Rad) and
blotted with primary monoclonal anti-GFP antibody
(CLONTECH) and secondary anti-mouse horseradish
peroxidase antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).
Peroxidase activity was developed with ECL (Amersham Pharmacia Biotech).
Confocal Microscopy--
To investigate localization of GFP and
GFP-fusion proteins, cells were transferred to a mounting chamber on
the microscope stage in Dulbecco's modified Eagle's medium/F12 medium
supplemented with 15 mM HEPES and 5% fetal bovine serum at
pH 7.2 after 66 h of transfection. Subsequently, cells were loaded
with 40 nM MitoTracker Red CMXRos (23) (Molecular Probes
Inc., Eugene, OR) for 15 min in culture medium. A 63X N.A. 1.4 oil
immersion planapochromat objective with a Zeiss 410 confocal microscope (Thornwood, NY) was used for all experiments. Confocal images of green
cytochrome c-GFP and GFP fluorescence were collected using
488 nm excitation light from an argon/krypton laser, a 560-nm dichroic
mirror and a 500-550-nm band pass barrier filter. Images of red
MitoTrackerRed CMXRos fluorescence were collected using a 568-nm
excitation light from the argon/krypton laser, a 560-nm dichroic
mirror, and a 590-nm long pass filter.
To measure mitochondrial membrane potential, cytochrome
c-GFP transfected PC6 cells were loaded with 150 nM TMRM for 15 min in culture medium (24). Subsequently,
cells were washed twice with fresh medium and transferred to a mounting
chamber on the microscope stage in medium containing 50 nM
TMRM. Temperature of the chamber was maintained at 35-37 °C with a
heat controller (Bioptechs, Butler, PA). Images of green cytochrome
c-GFP fluorescence were collected as described above and red
TMRM fluorescence was imaged using same filter settings as for
MitoTrackerRed CMXRos. For measurements of cytochrome c-GFP
and TMRM fluorescence, laser power was attenuated with neutral density
filters at least 90% and 99.9%, respectively. In addition, the images
were collected at 1 h intervals at the lowest zoom setting. These
precautions were taken to avoid photodamage caused by intense laser
illumination. After collecting basal images (0 h), 5 µM
staurosporine was added. Subsequently, images were collected at 2, 4, and 5 h. For the assessment of apoptotic cell nuclei, cytochrome
c-GFP-transfected cells were stained with 500 nM
SYTO-13 and imaged using the same filter settings as for cytochrome
c-GFP.
Rat PC6 cells were transiently transfected with plasmids bearing
the cytochrome c-GFP fusion construct. To confirm that
cytochrome c-GFP was targeted to mitochondria, cells were
co-loaded with Mitotracker Red CMXRos, which localizes to mitochondria
(23). Cytochrome c-GFP and Mitotracker Red CMXRos were
imaged using laser scanning confocal microscopy. As shown in Fig.
1A, cytochrome c-GFP displayed a punctate pattern of fluorescence that was
indistinguishable from that of MitoTracker Red. These confocal images
demonstrated that the cytochrome c-GFP fusion protein was
localized to mitochondria (Fig. 1, A and A'). By
contrast, cells transfected with GFP cDNA alone displayed a diffuse
fluorescence (Fig. 1B). The expression of the cytochrome
c-GFP fusion protein was also confirmed by Western blot
analysis using anti-GFP antibody (21, 22) (Fig. 1C).
INTRODUCTION
Top
Abstract
Introduction
References
) was monitored by rhodamine 123 and
DiOC6, compounds that are known to inhibit the ATP synthase
and mitochondrial respiration (14-16). In addition, cells respond
asynchronously to toxic insults (17-19), and assessment of sequential
events that occur rapidly becomes difficult when a population of cells
rather than individual cells is studied.
, we used
tetramethylrhodamine methyl ester (TMRM). TMRM has no
apparent effect on metabolism, and it distributes into
mitochondria in response to membrane potential without covalent binding
(20). To monitor cytochrome c, we made a cytochrome
c-green fluorescent protein (GFP) fusion construct for
transfection so that release of cytochrome c could be
observed in individual mitochondria by confocal microscopy (21). In the present report we show that mitochondrial depolarization is
accompanied by cytochrome c release in
staurosporine-induced apoptosis.
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Fig. 1.
Cytochrome c-GFP localizes
to mitochondria in PC6 cells. PC6 cells were transfected with
cytochrome c-GFP (A and A') and GFP
cDNA (B and B'). Cells were loaded with 40 nM MitoTracker Red CMXRos. Green fluorescence of cytochrome
c-GFP (A) and GFP (B) and red
fluorescence of MitoTrackerRed CMXRos (A and B')
was visualized by confocal microscopy. Cytochrome c-GFP
displayed a punctate pattern of fluorescence (A) that
matches that of MitoTracker Red CMXRos (A'), whereas GFP
fluorescence (B) was diffuse and did not match MitoTracker
Red (B'). Scale bar, 10 µm. C, Western blot of
cytochrome c-GFP and GFP. In cells transfected with the
cytochrome c-GFP construct, expression of the expected
43-kDa fusion protein band was revealed. By contrast, transfection of
native GFP cDNA led to expression of a 30-kDa protein, which is
close to the molecular weight of GFP.
To monitor changes in mitochondrial , cytochrome
c-GFP-transfected cells were loaded with TMRM. TMRM is a
cationic fluorophore that accumulates electrophoretically into
mitochondria in response to the negative mitochondrial (20).
When mitochondria depolarize, they release TMRM and the red
fluorescence inside mitochondria disappears. Accordingly, the green
fluorescence of cytochrome c-GFP and the red fluorescence of
TMRM were monitored simultaneously in single living cells by laser
scanning confocal microscopy. Fig.
2A shows a field of PC6 cells
transfected with cytochrome c-GFP (right panel)
and loaded with TMRM (left panel). Before addition of
staurosporine to induce apoptosis, all cells in the field displayed
high mitochondrial , as indicated by bright punctate fluorescence
of TMRM (Fig. 2A, 0 h, left panel). Not all
cells were transfected with cytochrome c-GFP, as indicated by the absence of green fluorescence, but all viable cells had polarized mitochondria as revealed by red fluorescence (Fig.
2A, 0 h, left panel).
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To induce apoptosis, cells were treated with 5 µM staurosporine, a protein kinase inhibitor, which induces apoptosis in a wide variety of cell types (12, 17, 25, 26). In the field of PC6 cells shown in Fig. 2A, TMRM fluorescence began to decrease in cell 1 after 2 h (Fig. 2A, 2 h, left panel). The decrease of fluorescence was because of the disappearance of a portion of the bright fluorescent mitochondria indicating that some but not all mitochondria had depolarized and released their TMRM. Simultaneously, the pattern of cytochrome c-GFP fluorescence began to change from punctate to diffuse, indicating release of cytochrome c-GFP from the mitochondria (Fig. 2A, 2 h, cell 1, right panel). As cytochrome c-GFP was released, green fluorescence increased diffusely in the cytosol and in the nucleus as well. At this early stage, cytochrome c-GFP fluorescence was not fully diffuse, and a punctate cytochrome c-GFP pattern remained in mitochondria that still retained TMRM fluorescence. After 3 h, however, all mitochondria depolarized, as indicated by the disappearance of virtually all TMRM fluorescence (data not shown). By this time cytochrome c-GFP fluorescence disappeared virtually completely from cell 1, which likely indicated onset of the secondary necrosis that often follows apoptosis. At the onset of cell necrosis, the plasma membrane ruptures and soluble cytosolic proteins, such as cytochrome c-GFP, leak out. However, apoptosis was the primary mode of cell death in the cells studied, as indicated by nuclear shrinkage, chromatin condensation, and formation of apoptotic bodies assessed by the nuclear stain SYTO-13 (Fig. 2B, Stauro).
Similarly, in cell 2 the number of TMRM-labeled mitochondria began to decrease after 2 h of exposure to staurosporine (Fig. 2A, 2 h, cell 2, left panel). At this time, the pattern of cytochrome c-GFP fluorescence was unchanged (Fig. 2A, 2 h, cell 2, right panel). Subsequently, after 4 h, a greater portion of mitochondria released their TMRM fluorescence (Fig. 2A, 4 h, cell 2, left panel), and cytochrome c-GFP fluorescence pattern changed from punctate to partially diffuse, indicating a partial redistribution of cytochrome c-GFP from mitochondria to the cytosol and nucleus (Fig. 2A, 4 h, cell 2, right panel). After 5 h, all mitochondria in cell 2 had released their TMRM, and cytochrome c-GFP fluorescence showed a fully diffuse pattern. All the cytochrome c-GFP transfected cells in the field behaved asynchronously regarding onsets of mitochondrial depolarization and cytochrome c release.
To confirm that the changes we observed regarding mitochondrial
depolarization and cytochrome c-GFP fluorescence were indeed related to staurosporine treatment, we observed cells without staurosporine. Mitochondrial and cytochrome c-GFP
distribution did not change in the absence of staurosporine (Fig.
2C). Also the nuclear morphology of the cells remained
normal (Fig. 2B, Cont). These results excluded the
possibility that mitochondrial depolarization and cytochrome
c-GFP redistribution would be related to photodamage induced
by laser (27).
To quantitate the changes in TMRM fluorescence over time, we measured total TMRM fluorescence in each cell. Fig. 3A shows total TMRM fluorescence (x ± S.E.) of all cells studied. After staurosporine, TMRM fluorescence decreased 50% in 5 h. Untreated cells showed no decline in TMRM fluorescence. Fig. 3B plots individual cellular TMRM fluorescence for each of the cells shown in Fig. 2A. All cells, except cell 7, showed 10-40% decline in TMRM fluorescence after 2 h of exposure to staurosporine. Subsequently, TMRM fluorescence declined further in each cell in a variable manner. After 7 h, five of nine cells displayed no TMRM fluorescence. Because images of these same cells revealed that individual mitochondria released their TMRM fluorescence heterogeneously in an all-or-none fashion (Fig. 2A), the decrease of total TMRM fluorescence in individual cells does not mean that all mitochondria within a cell were partially depolarized (Fig. 3B). Rather, a proportion of mitochondria within a cell were fully depolarized. Because the depolarized mitochondria had also lost their punctate cytochrome c-GFP fluorescence, our results indicate strongly that mitochondrial depolarization accompanied and probably preceded cytochrome c-GFP release.
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Our fusion construct is a 43-kDa protein, whereas endogenous cytochrome c is a 15-kDa protein. Thus, endogenous cytochrome c might be released before mitochondrial depolarization and before release of the larger molecular weight fusion protein. To exclude this possibility, we monitored the release of endogenous cytochrome c in staurosporine-treated cells by immunocytochemistry (28). Like cytochrome c-GFP, endogenous cytochrome c displayed punctate fluorescence both in untreated cells and in cells treated with staurosporine for 1 h. After longer exposure to staurosporine, cytochrome c staining progressively became more diffuse (data not shown).
Our results show directly for the first time that mitochondrial depolarization and cytochrome c release occur simultaneously in individual mitochondria of single living cells during staurosporine-induced apoptosis. Although the present work does not attempt to define the mechanism of cytochrome c release from mitochondria to the cytosol, the results are compatible with the sequence of events proposed in the MPT hypothesis. Exposure of cells to staurosporine may cause the onset of MPT followed by mitochondrial depolarization. These events may result in matrix swelling because of uptake of water and solutes via the MPT. The expansion of matrix may rupture the mitochondrial outer membrane leading to the release of cytochrome c and other caspase-activating proteins from the intermembrane space (10, 29-31).
When cytochrome c-GFP was released from the mitochondria
into the cytosol, green fluorescence increased in the nucleus in all
cells studied. In PC6 cells, the cytosol is a small proportion of cell
volume that is mostly comprised of nucleoplasm. Once released from
mitochondria into the cytosol, cytochrome c-GFP rapidly
diffused into the nucleus, presumably through the nuclear pores. Thus, increases of nuclear cytochrome c-GFP reflected increases of
cytosolic cytochrome c-GFP. The importance of this nuclear
cytochrome c-GFP in the apoptotic process remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank R. C. Scarpulla from Northwestern University Medical School, Chicago for cytochrome c cDNA, F. W. Holtszberg from University of Kentucky, Lexington for PC6 cell line, and J. J. Lemasters and J. C. LaManna for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported in part by Grant AG 13318 from National Institutes of Health and a Grant-in-Aid from the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the American Heart Association Northeast Ohio Affiliate and the Academy of Finland.
** To whom correspondence should be addressed: Dept. of Anatomy, Case Western Reserve University, School of Medicine W520, 10900 Euclid Ave., Cleveland, OH 44106-4930. Tel.: 216-368-0069; Fax: 216-368-1144; E-mail: axn25{at}po.cwru.edu.
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ABBREVIATIONS |
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The abbreviations used are:
MPT, mitochondrial
permeability transition;
PC6, pheochromocytoma-6;
, membrane
potential;
TMRM, tetramethylrhodamine methyl ester;
GFP, green
fluorescent protein.
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REFERENCES |
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Introduction
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