* Biochemistry Section, Bax, a member of the Bcl-2 protein family,
accelerates apoptosis by an unknown mechanism. Bax
has been recently reported to be an integral membrane
protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused
to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal
microscopy performed on living Cos-7 kidney epithelial
cells and L929 fibroblasts revealed that GFP-Bcl-2 and
GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax
was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble
protein, in contrast to organelle-bound GFP-Bcl-2. The
diffuse localization of GFP-Bax did not change with
coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially
colocalized with mitochondria. Once initiated, this Bax
movement was complete within 30 min, before cellular
shrinkage or nuclear condensation. Removal of a
COOH-terminal hydrophobic domain from GFP-Bax
inhibited redistribution during apoptosis and inhibited
the death-promoting activity of both Bax and GFP-
Bax. These results demonstrate that in cells undergoing
apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to
promote cell death.
PROGRAMMED cell death is vital for both normal development and maintenance of many tissues in multicellular organisms. A form of programmed cell
death, apoptosis, proceeds with morphological and biochemical characteristics that include blebbing of the cell membrane, a decrease in cell volume, nuclear condensation, and the intranucleosomal cleavage of DNA (37).
The family of Bcl-2-related proteins plays key roles in
the regulation of apoptosis. Individual family members can
function to either block or promote programmed cell
death (38). Three extensively characterized mammalian
family members are the antiapoptotic genes bcl-2 and bcl-XL, and the proapoptotic gene bax. In vivo studies suggest
that a single protein product predominates for each gene:
Bcl-2 Previous investigations of Bax localization have relied
upon cell fixation or fractionation. In this study, we examine Bax localization in living cells. The genes encoding human Bcl-2, Bcl-XL, and Bax were fused to that of the green
fluorescent protein (GFP),1 and herein we report the localization by confocal microscopy of these fusion proteins
in both healthy cells and cells undergoing apoptosis. The
time course of changes in GFP-Bax localization relative to
other cellular events occurring during apoptosis was characterized. Furthermore, the influence of Bcl-2 and Bcl-XL overexpression on Bax localization was examined. We
have deleted the COOH-terminal hydrophobic tails from
Bax, Bcl-2, and Bcl-XL to investigate the importance of
these regions in the subcellular localization, and the ability
of these proteins to influence cell death.
Except as noted, reagents were purchased from Sigma Chemical Co. (St.
Louis, MO).
Generation of Expression Constructs
Full-length and COOH-terminal truncated forms ( Cell Culture and Transient Transfection
L929 murine fibrosarcoma and Cos-7 green monkey renal epithelia cell
lines (American Type Culture Collection, Rockville, MD) were grown in
Minimum essential medium, Eagle's in Earle's balanced salt solution and
DME, respectively, each supplemented with 2 mM glutamine, 1× nonessential amino acids, 2.5 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/
ml streptomycin (all from Biofluids Inc., Rockville, MD), 50 µg/ml gentamicin (GIBCO BRL), and 10% heat-treated FCS. Cells were cultured at
37°C in 5% CO2 and then split twice weekly using 0.05% trypsin/0.02%
versene (Biofluids Inc.). In preparation for transfection, cells were plated
at a density of 2 × 105 cells per well in six-well tissue-culture plates (Becton Dickinson Labware, Bedford, MA). Either one (Cos-7) or two (L929)
days later, cells were transiently transfected using the cationic lipid LipofectAmine (GIBCO BRL) as described by the manufacturer, using 2 µg
of plasmid DNA and either 8 (L929) or 10 µl (Cos-7) of LipofectAmine
per well. After 5 h in serum-free medium, an equal volume of medium containing 2× normal FCS was added. 1 d after transfection, the cells
were washed once and placed in normal growth media. Transfected cells
were then cultured for an additional 24 h, and visualized by microscopy.
For cotransfection experiments (see Fig. 7), the above procedure was followed, except that a total of 4 µg of plasmid DNA was used per well: 1 of
C3-EGFP-Bax and 3 of either pcDNA3-Bcl-2 or pcDNA3-Bcl-XL.
Generation of Anti-human Bcl-2 Monoclonal Antibody
A peptide corresponding to amino acids 41-54 of human Bcl-2 (GAAPAPGIFSSQPGC; Peptide Technologies Corp., Gaithersburg, MD) was conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) (Pierce
Chemical Co., Rockford, IL) through the cysteine residue according to the
protocol provided by the manufacturer. Mice were immunized with the
peptide-KLH conjugate and splenocytes from immunoreactive mice were
fused by PEG 4,000 to murine NS-1 myeloma cells and selected with histone acetyltransferase (HAT) medium as previously described (15). The
anti-human Bcl-2 antibody was designated Cell Lysis and Western Blotting
Transfected cells from two wells of a six-well plate were washed once with
PBS and then lysed in cold solubilization buffer (PBS, 25 µg/ml phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin) containing 1% Triton X-100. For Western blotting, 20 µl of each sample were
separated by electrophoresis using 12% SDS-PAGE gels. Gels were electrotransferred onto Immobilon-P (Millipore Corp., Waters Chromatography, Milford, MA) membranes. The blots were blocked in PBS/0.05%
Tween 20 containing 5% fetal bovine serum and incubated with the appropriate antibody: anti-human Bcl-2 8C8, anti-universal Bcl-XL 2H12
(15), or anti-human Bax 2D2 monoclonal antibodies (15). Primary antibody binding was detected by blotting with sheep anti-mouse F(ab')
linked to horseradish peroxidase (Amersham Corp., Arlington Heights,
IL), followed by band visualization using enhanced chemiluminescence as
described by the manufacturer (Amersham Corp.).
Confocal Microscopy
Cells used for confocal microscopy were transfected as described above
on 1.5 thickness glass coverslips pretreated with 5 µg/ml poly-L-lysine. After 24-48 h, cells were prepared for microscopy by incubation with 20 ng/
ml of a mitochondrion-specific dye (Mitotracker Red CMXRos; Molecular Probes Inc., Eugene, OR) and/or 100 ng/ml bis-benzamide for 30 min,
as indicated in individual experiments. After pretreatment, cells were
transferred to a sealed mounting chamber containing fresh medium; in indicated experiments, this medium was supplemented with 1-3 µM staurosporine (STS). Images were collected on a microscope (model LSM 410 with a 40× 1.2 NA Apochromat objective; Carl Zeiss, Thornwood, NY).
The 488- and 568-nm lines of a krypton/argon laser were used for fluorescence excitation of GFP and Mitotracker Red CMXRos, respectively, and the 364-nm line of an argon laser was used for excitation of bis-benzamide dye. The temperature of the specimen was maintained between 35° and
37°C with an air stream incubator.
To investigate the mobility of the GFP-fusion proteins, the fluorescence within a region of a cell was photobleached by scanning the region
with the 488-nm laser line at full power (no attenuation) and then the entire cell was scanned at 10-s intervals with low laser power (10% power,
0.3% attenuation) to monitor the recovery of fluorescence. The fraction
of the fluorescent protein that was mobile was calculated as described (4)
by comparing the fluorescence intensities in two regions of the cell, one
inside the photobleach zone and the other elsewhere, before and 100 s after the photobleach (mobile fraction = ratio after 100-s recovery/ratio before photobleach). The time course of fluorescence recovery was determined by photobleaching a 16-µm strip within a flattened area of the cell
and then monitoring the recovery of fluorescence within the strip (200 images at 0.46-s intervals).
Cell Viability Assay
To assess the effects of the full-length and GFP-Bax Is a Cytosolic Protein in Healthy Cells
Whereas GFP-Bcl-2 and the Majority of Bcl-XL
Associate with Mitochondria
We examined the location of Bcl-2, Bcl-XL, and Bax in individual living cells by using GFP fusion constructs. Fusion
constructs containing GFP have been successfully used to
study the subcellular localization of a number of proteins
(16, 26, 33). The gene encoding a high fluorescence, red-shifted GFP variant (5) was fused to the 5 The localization of these transiently expressed GFP-
linked fusion proteins was visualized in living cells using
confocal microscopy. In both Cos-7 (Fig. 1, a and b) and
L929 cells (Fig. 2, a and b), GFP alone has a diffuse distribution, filling the entire cell as previously reported (28).
GFP-Bcl-2 displays a punctate pattern of distribution, primarily in areas near the nucleus, in both Cos-7 (Fig. 1, e
and f) and L929 (Fig. 2, e and f) cell lines. This distribution
matches the localization determined previously for Bcl-2
by immunofluorescence (24, 41), and suggests association
with an intracellular organelle or organelles. GFP-Bcl-XL displays a bright punctate distribution similar to that of GFP- Bcl-2 superimposed upon a fainter diffuse background
(Figs. 1 and 2, i and j), suggesting both soluble and membrane-bound populations in individual cells. This is consistent with the dual localization of Bcl-XL in both the soluble
and membrane pellets in cellular fractionation experiments
(14, 15). In contrast to the localization of GFP-Bcl-2 and
GFP-Bcl-XL, GFP-Bax has a diffuse distribution similar
to that of GFP alone in both Cos-7 (Fig. 1, m and n) and L929 (Fig. 2, m and n) cell types.
We examined the GFP fusion protein expression by
Western blot analysis of transiently transfected Cos-7 cells
36 h after transfection. Immunoblotting of these lysates
confirmed that GFP-Bcl-2 (Fig. 3, middle, arrow a), GFP-
Bcl-XL (Fig. 3, bottom, arrow a), and GFP-Bax (Fig. 3,
top, arrow a) are of appropriate size. Low molecular weight bands (lane c) representing native Bcl-2, Bcl-XL,
and Bax were detected in all lysates. An additional, unexpected protein band (arrow b) of intermediate molecular
weight was detected for each expression construct (Fig. 3).
This intermediate molecular weight form was also found
when COOH-terminal truncated forms of Bcl-2 (lane c),
Bcl-XL (lane e), and Bax (lane a) fused to GFP were expressed and immunoblotted. When compared to full-length forms, all three COOH-terminal truncated proteins ran at
the expected slightly lower molecular weights (Fig. 3). The
COOH-terminal truncated constructs display slightly lower
molecular weights for both the high molecular weight and
intermediate molecular weight bands, indicating that the
intermediate weight bands likely represent proteins shortened from the NH2-terminal, GFP end, of the fusion protein. Thus, the intermediate size bands may result from expression of transcripts initiated at an internal start codon
within the GFP gene or proteolytic fragments lacking part
of the GFP molecule, and thus they may or may not fluoresce.
To determine if the fusion of GFP to Bcl-2, Bcl-XL, and
Bax affected their bioactivities, we compared the viability
of either L929 or Cos-7 cells transfected with wild-type
cDNA constructs to those transfected with GFP-fusion constructs after induction of apoptosis with STS. As shown in
Fig. 4, the protective activities of GFP-Bcl-2 (Fig. 4 A) and
GFP-Bcl-XL (Fig. 4 B) in L929 cells are comparable to those
of the wild-type forms. Likewise, the activity of GFP-Bax
appears to accelerate cell death of Cos-7 cells as effectively as does wild-type Bax (Fig. 4 C). Thus, adding GFP
to the NH2-terminus of Bcl-2, Bcl-XL, or Bax does not
block their abilities to influence apoptosis. However, the
addition of a large protein to the NH2 terminus may alter
the potency of Bcl-2 family members to regulate apoptosis
or influence their intracellular localization.
To investigate the identity of the organelle or organelles
to which the GFP-Bcl-2 and the majority of the GFP-Bcl-XL
localize, we treated transiently transfected Cos-7 cells with
Mitotracker Red CMXRos. As shown in Fig. 5 (a-c), the
pattern of GFP-Bcl-2 distribution coincides very closely
with that of the mitochondrial marker, demonstrating that
most of the GFP-Bcl-2 is found associated with mitochondria. The punctate fraction of the GFP-Bcl-XL also distributes to mitochondria (Fig. 5, d-f). These results match earlier findings localizing Bcl-2 and Bcl-XL to the membrane of mitochondria using other imaging modalities (19,
24). In constrast to GFP-Bcl-2 and GFP-Bcl-XL, GFP-
Bax does not localize to mitochondria in healthy Cos-7
cells (Fig. 5, g-i).
FRAP Indicates Bcl-2 Is Largely Immobile Whereas
Bax Diffuses Freely
The similarity of the distribution of GFP-Bax to the distribution of GFP led us to investigate whether GFP-Bax is
free to diffuse within the cytoplasm, as GFP has been shown
to be (34). The mobilities of GFP-Bax and GFP were compared by photobleaching the fluorescence in a small region of the cell and then monitoring the recovery of fluorescence due to movement of unbleached molecules into
the bleached zone. Both GFP and GFP-Bax moved rapidly into the bleached zone (Fig. 6). The time courses for
fluorescence recovery of GFP and GFP-Bax were essentially identical (Fig. 6 B), and were consistent with diffusion kinetics. The proteins diffused too rapidly relative to
the rates at which we could photobleach and monitor recovery to allow us to accurately determine diffusion constants.
Photobleaching experiments also were performed on cells
expressing GFP-Bcl-2 (Fig. 6). In contrast to GFP and GFP-Bax, GFP-Bcl-2 appeared to be largely immobile. The mobile
fraction (percentage recovery) at 100 s after photobleaching (calculated as described in Materials and Methods) was
9 to 13% for GFP-Bcl-2 (n = 3) as compared with 45 to
85% for GFP-Bax (n = 4), and 82 ± 2% for GFP alone
(34). The low mobility of GFP-Bcl-2 supports the conclusion that it resides in discrete organelles (mitochondria)
rather than in a continuous membrane system such as the
endoplasmic reticulum (where fluorescence recovers rapidly after photobleaching [4]).
Role of Bcl-2 in Docking Bax
Recent studies have proposed that Bax (32) and Bad (40)
require Bcl-2 to associate with organelle membranes. To test
the possibility that GFP-Bax localization to organelles is
limited by the endogenous level of either Bcl-2 or Bcl-XL,
vectors expressing either Bcl-2 or Bcl-XL were cotransfected with the GFP-Bax vector. As shown in Fig. 7 A, coexpression of Bcl-2 at high levels, verified by immunoblotting of cell lysates (Fig. 7 B), did not alter the cytoplasmic
distribution of GFP-Bax. Similar results were obtained when Bcl-XL was coexpressed with GFP-Bax (data not
shown). Thus, we find Bax is a soluble, cytosolic protein in
healthy cells, which does not bind organelles even in the
presence of elevated levels of Bcl-2 or Bcl-XL.
Bax Localization and Mobility Change during Apoptosis
Endogenous Bax and some of the endogeneous Bcl-XL
migrate from the supernatant to the membrane pellets in
cell fractionation experiments performed on thymocytes
undergoing apoptosis (14). To investigate this phenomenon in living cells, Cos-7 cells were transiently transfected
with native GFP, GFP-Bcl-2, GFP-Bcl-XL, and GFP-Bax.
Cells expressing each construct were treated with 1 µM
STS, and after 6 h both STS-treated and untreated cells
were examined by confocal microscopy. STS-treated cells
underwent cell shrinkage and blebbing of the cell membrane, a morphology indicative of apoptosis (Fig. 1, compare b, f, j, and n with d, h, l, and p). GFP-Bax markedly
changed its intracellular distribution, relocating within cells
during apoptosis from a diffuse (Fig. 1 m) to a punctate pattern (Fig. 1 o). This change was not observed with GFP
alone (Fig. 1 c). No changes in the patterns of distribution
of either GFP-Bcl-2 (Fig. 1 g) or GFP-Bcl-XL (Fig. 1 k)
were observed in treated cells beyond those related to
changes in cell size and shape. It is possible that redistribution of soluble GFP-Bcl-XL is reduced to some extent in
this system due to the ability of overexpressed GFP-Bcl-XL
to inhibit apoptosis (Fig. 4 B). Similiar experiments performed using another cell line, L929 fibroblasts, confirmed
that GFP-Bax undergoes a shift from a diffuse to punctate distribution pattern during apoptosis whereas the localization patterns of GFP-Bcl-2 and GFP-Bcl-XL remain essentially unchanged (Fig. 2).
Mitochondrial staining of GFP-Bax-expressing Cos-7 cells
undergoing apoptosis suggests that much of the GFP-Bax
attaches to or enters mitochondria (Fig. 8, a-c). However,
as there are also sites of Bax condensation that do not
stain for mitochondria, it is possible that GFP-Bax is also
localizing to other organelles upon induction of apoptosis
or that mitochondria lose their Mitotracker Red CMXRos
staining during apoptosis.
The redistribution of Bax to organelles induced by STS
treatment is associated with a reduction in Bax mobility
(Fig. 6). FRAP experiments on cells in which GFP-Bax had
a punctate (mitochondrial) localization indicated that the
mobile fraction was 10 to 30% (n = 2), as compared with
45 to 85% in untreated cells. Thus, the punctate form of
GFP-Bax is less mobile than the diffuse form of GFP-Bax
consistent with the model that Bax redistributes from a
cytoplasmic to membrane-bound localization during apoptosis.
Time Course of Bax Redistribution during Apoptosis
To relate the timing of the shift in GFP-Bax localization
to other morphological events that occur during apoptosis,
individual Cos-7 cells expressing GFP-Bax were followed
by confocal microscopy over the course of STS-induced
cell death. The change in GFP-Bax localization within individual cells occurred swiftly, over the course of ~30 min
after initiation (Fig. 9, c-e). Various cells in a given field
may begin the process of GFP-Bax redistribution at different times, as seen in Fig. 9. At the time point when GFP-Bax begins redistribution, little alteration in the overall structure of the cell is evident when viewed by differential interference contrast (DIC) microscopy (Fig. 9 h), or
by interference reflection microscopy that shows the cell-
substrate interaction (data not shown). At 10-30 min after
the initiation of Bax redistribution, cell shrinkage first became apparent by DIC imaging (Fig. 9, i and j, arrows).
Cells expressing GFP-Bax were incubated with the nuclear stain bis-benzamide (Hoechst 33342), which labels
DNA and is commonly used to detect the nuclear changes
characteristic of apoptosis. At low concentrations, this stain
can be used to study the location of DNA within living and
even dividing cells without affecting cell viability (8). After
bis-benzamide staining, cells were then treated with STS
to induce apoptosis. As shown in Fig. 10, there is no evidence of nuclear changes when GFP-Bax initially begins
redistribution. At ~3 h after the initiation of the GFP-Bax transition, a more uneven pattern of bis-benzamide staining can be seen in the nuclei of cells (Fig. 10 e), suggestive
of nuclear condensation. This gradually progresses to nuclear fragmentation, clearly apparent by 12 h after initiation of GFP-Bax redistribution (Fig. 10 f). Therefore, the
shift in GFP-Bax distribution appears to be an early event
in apoptosis, preceding several established morphological
changes associated with apoptosis.
COOH-terminal Hydrophobic Region Is Required for
Bax Redistribution
The COOH-terminal hydrophobic region, spanning the last
20-24 amino acids of Bcl-2, Bcl-XL, and Bax is thought to
act as an anchor localizing these proteins to the membranes
of organelles. As our results suggest that Bax localizes to
organelles only upon induction of apoptosis, we investigated the role of the COOH terminus in Bax trafficking.
Deletion of the COOH-terminal Tail Attenuates the
Influence of Bcl-2, Bcl-XL, and Bax on Cell Viability
If Bax insertion into organelles is required for death promoting activity, then Bax Bax promotes programmed cell death in mammalian cells
(18, 27). However, Bax expression per se, at least at physiological levels, is not lethal to cells. For example, sympathetic neurons express high levels of bax mRNA, yet will
not undergo apoptosis unless deprived of growth factors
(6). As initially characterized, even cells stably overexpressing Bax proliferate normally (27), and Bax only accelerates cell death after an external signal, such as interleukin-3 withdrawal. As protein levels of Bax do not
change during apoptosis (14, 27), it seems plausible that a
posttranslational activation of Bax occurs during apoptosis. Our findings indicate that Bax activity may be regulated at the level of intracellular localization, with Bax
moving to the membranes of organelles after induction of
apoptosis, but before nuclear fragmentation. Elimination
of 21 amino acids from the COOH terminus prevents Bax
redistribution and abrogates Bax apoptosis-promoting activity, correlating organelle binding with bioactivity.
In contrast to cytosolic GFP-Bax, GFP-Bcl-2 displays a
punctate distribution in healthy cells that coincides very
strongly with a mitochondrial marker. GFP-Bcl-XL demonstrates a mixed pattern, suggesting that although much
of the protein is associated with mitochondria, a significant
amount is found diffusely throughout the cytosol. In cell
fractionation studies, this soluble Bcl-XL found in healthy
cells redistributes to associate with the membrane during
apoptosis (14). No change in distribution is observed for
either GFP-Bcl-2 or GFP-Bcl-XL after the addition of STS. However, the levels of GFP-Bcl-XL in the transfection experiments are sufficient to delay or even block apoptosis
(Fig. 4); thus, GFP-Bcl-XL may actually prevent alterations in the intracellular milieu that would lead to its redistribution. Further experiments will be necessary to explore the intracellular distribution of GFP-Bcl-XL during
apoptosis, perhaps by using apoptosis pathways that are
Bcl-XL insensitive.
For all three of the Bcl-2 family members examined here,
the COOH-terminal deletion eliminates the ability of GFP-tagged proteins to associate with organelles. The dependency of Bax redistribution on the COOH-terminal hydrophobic domain suggests that a conformational change
or other modification might occur in Bax during apoptosis, allowing the previously inaccessible or blocked hydrophobic tail to insert into membranes. It is striking that the
translocation domain of diphtheria toxin, which has structural homology to Bcl-XL, displays a similar ability to
change from a soluble to membrane-associated state. In
the case of the diphtheria toxin translocation domain, this
insertion is driven by a conformational change triggered by low pH (7, 30). It remains unclear how Bax insertion
into membranes is controlled. One interesting candidate is
phosphorylation: a regulatory mechanism involving phosphorylation has recently been described that influences
both localization and activity of the apoptosis promoter
Bad (40), and phosphorylation of Bcl-2 has been suggested
to regulate its function (10, 17, 22).
Our findings conflict with previous reports that suggest
that Bax is constitutively localized to organelle membranes
(for review see reference 29). One possible explanation for
this discrepancy may be that in earlier work, Bax localization was examined in contexts where overexpression of
Bax alone was sufficient to cause apoptosis, without the
overt induction of apoptosis. In a study examining Bax localization in a transformed BRK cell line, Bax overexpression led directly to cell death (11). A recent paper on Bax
localization to mitochondria (39) reported that the expression of fusion proteins containing full-length Bax was lethal
to yeast cells, and removal of the COOH terminus abolished the ability of Bax to kill yeast cells and localize to yeast
mitochondria. We have found that cationic lipid transfection procedures can themselves trigger apoptosis (Wolter,
K.G., and X.-G. Xi, unpublished observations). Deletion
of the COOH-terminal tail from GFP-Bax eliminated this
increase in transfection-associated death. High levels of
full-length GFP-Bax expression may, therefore, lead directly to the death of transfected cells, particularly if this
expression closely follows the cellular stress of the transfection procedure. Perhaps some previous observations of organelle-located Bax were made on cells entering an apoptosis program. These reports and our own data concur in
suggesting that Bax localization to organelles is a vital aspect of its death promoting ability.
However, our findings on the role of Bcl-2 in Bax localization clearly differ from those of Shibasaki and co-workers (32), who observed a redistribution of soluble Bax to a
punctate pattern in BHK cells expressing high levels of
Bcl-2. In our systems, we see no evidence for a localization
of Bax to membranes driven by either Bcl-2 or Bcl-XL coexpression. This is consistent with earlier findings that Bax
and Bcl-XL do not form heterodimers in healthy cells (15).
Recent reports indicate that Bcl-2 family members control the release of cytochrome C and apoptosis-inducing
factor from mitochondria during apoptosis and contribute
to cell death by caspase activation (20, 21). The overexpression of Bcl-2 has been reported to block the mitochondrial release of cytochrome C, and in doing so prevents
apoptosis (38). These results are interesting, given the structural connection made between Bcl-XL and the diphtheria toxin translocation domain. This domain is capable of forming transmembrane ion channels, and is furthermore believed to multimerize to form pores that mediate the translocation of the catalytic domain of the diphtheria toxin across
membranes. Bcl-XL and Bcl-2 have been reported to form
transmembrane channels (23, 31). In this context, it is possible that upon Bax insertion into membranes, Bax may
form channels or pores allowing for the release of factors such as cytochrome C from within the mitochondria to
propagate the apoptotic pathway. This model does not preclude the interaction of Bax with Bcl-2 and Bcl-XL upon
membrane insertion. In the future, it will be crucial to elucidate the trigger regulating Bax insertion into organelle
membranes and the molecular consequences of membrane-bound Bax.
Received for publication 15 July 1997 and in revised form 12 September
1997.
We would like to thank C. Croce, C. Thompson and S. Korsmeyer for the
gifts of Bcl-2, Bcl-XL, and Bax genes, respectively. We thank R. Kocher, J. Castelli, X.-H. Liu, C. Ng, and P. Johnson for assistance, and L. Marrone
(all from National Institutes of Health [NIH], Bethesda, MD), and K. Wood (Trevigen Inc., Gaithersburg, MD) for reading the manuscript.
Light Microscopy Facility, Laboratory of Molecular Biology, National
Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892
Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References
Abstract
, a 26-kD protein (36); Bcl-XL, a 27-kD protein (9);
and Bax
, a 21-kD protein (27). (Bcl-2
and Bax
hereafter will be referred to as Bcl-2 and Bax.) Bcl-2, Bcl-XL, and
Bax each contain a stretch of hydrophobic amino acids,
~20 residues in length, at their COOH termini. There is
little amino acid sequence conservation within these tails,
but based on hydropathy plot analysis they are presumed
to function in anchoring these proteins into organelle membranes (2, 25). Bcl-2 has been localized to the nuclear
membrane, endoplasmic reticulum, and the outer mitochondrial membranes (3, 12, 19, 24). Removal of the COOH-terminal tail from Bcl-2 changes the subcellular
localization to the cytosol, but it remains unclear what effect the removal of the tail has on the ability of Bcl-2 to inhibit apoptosis (1, 2, 13, 35). Bcl-XL also has been localized
to the outer membrane of mitochondria (9). Although the
localization of Bax has been less extensively investigated,
Bax has been suggested to be targeted to organelle membranes (11), and in particular to mitochondria (39) by a
COOH-terminal hydrophobic region. However, it was recently reported that in BHK cells, low level expression of
Bax had a punctate, organelle localization whereas overexpressed Bax displayed a diffuse cytosolic distribution
when visualized by immunocytochemistry (32). In that
study, coexpression of high levels of Bcl-2 led to a punctate distribution of Bax, suggesting that Bax association
with organelle membranes required Bcl-2. In another recent study, the endogenous Bax in murine thymocytes, splenocytes, and HL-60 human leukemia cells was found
by subcellular fractionation to be predominantly soluble,
localized separately from membrane-bound Bcl-2 (14, 15).
During thymocyte apoptosis, a significant fraction of the
Bax was found to redistribute from the cytosol to membranes. This latter finding raises the possibility of the regulation of Bax subcellular localization during apoptosis (14).
Materials and Methods
CT) of human Bcl-2,
Bcl-XL, and Bax were synthesized by PCR amplification and cloned into
the pcDNA-3 mammalian expression vector (Invitrogen, Carlsbad, CA).
A similiar strategy was used for GFP-fusion constructs, using the C3-EGFP plasmid (Clontech Laboratories, Inc., Palo Alto, CA). The cDNAs
for human bcl-2, human bcl-XL, and human bax were gifts of C.M. Croee
(Jefferson Cancer Center, Philadelphia, PA), C.B. Thompson (HHMI and
University of Chicago, Chicago, IL), and S.J. Korsmeyer (HHMI and
Washington University, St. Louis, MO), respectively. The bcl-2
CT, bcl-XL
CT, and bax
CT forms constructed encode for proteins which have
a deletion of 23, 25, and 21 amino acids at their COOH-terminal ends, respectively. For full-length constructs of each gene, oligonucleotides placing a restriction enzyme site immediately adjacent to the start codon (forward primers) or stop codon (reverse primers) were synthesized (GIBCO
BRL, Gaithersburg, MD). For the truncated (
CT) forms, a stop codon
was introduced by the reverse primers. PCR was performed using Pfu
polymerase (Stratagene, La Jolla, CA) to minimize errors. PCR fragments
were purified, digested with restriction enzymes, and ligated into the expression vectors. The bcl-XL, bcl-XL
CT, bcl-2, and bcl-2
CT genes were
subcloned between the XhoI and XbaI sites in the polycloning regions of
both pcDNA3 and C3-EGFP. The bax and bax
CT genes were subcloned between the HindIII and EcoRI sites in the polycloning regions of
both pcDNA3 and C3-EGFP. All constructs were sequenced using the Sequenase 2 kit (United States Biochemical Corp., Cleveland, OH).
Fig. 7.
GFP-Bax distribution in Cos-7 cells is not affected by coexpression of
Bcl-2. Cells were transiently
transfected with a 3:1 ratio of
plasmid containing Bcl-2 and
GFP-Bax, and examined 48 h
later by confocal microscopy.
(A) In Cos-7 cells cotransfected with Bcl-2 and GFP-
Bax, the GFP-Bax distributes
throughout the cell in a pattern that is indistinguishable
from that of cells expressing
GFP-Bax alone. (B) The coexpression of GFP-Bax and human Bcl-2 in Cos-7 cells
was monitored by Western
blotting with human Bax
2D2 and
human Bcl-2 8C8
monoclonal antibodies. Lanes a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3-Bcl-2 transfection, and lanes c are from the cotransfection of C3-EGFP-Bax and pcDNA 3-Bcl-2. Arrows a, b, and c represent the GFP-Bax, its NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 µm.
[View Larger Versions of these Images (68 + 31K GIF file)]
hBcl-2 8C8.
CT forms of Bcl-2, Bcl-XL,
and Bax, on cell viability, a cotransfection assay was performed in L929 or
Cos-7 cells. Wells plated as described above were cotransfected with one
vector construct containing a given gene in pcDNA3, and the second vector, EGFP (Clontech Laboratories Inc.), containing a gene for a red-shifted GFP. A 3:1 molar ratio of the former to the latter was used, with a
total of 4 µg of DNA per well. Otherwise, cells were transfected as above.
At 36-48 h after transfection, cells were washed twice, fresh culture medium was added, and the number of GFP-positive cells in a defined field
was determined by examination with a fluorescence microscope. Apoptosis was then induced by the addition of medium containing STS at the concentrations indicated, and the number of GFP-positive cells within the same
field was assessed at regular intervals over the next 24-42 h. To test the effect on bioactivity of adding GFP to the NH2 termini of the Bcl-2 family members, a similar assay was performed on cells transfected with 2 µg of
the appropriate full-length or
CT gene fused to GFP inserted into C3-EGFP. For each construct, the assay was repeated three or four times. To
normalize for variations in transfection efficiency, changes in viability are
expressed as percentage decrease in the number of fluorescent cells within
a given field over time.
Results
termini of cDNAs
encoding human Bcl-2, Bcl-XL, or Bax. L929 murine fibrosarcoma and Cos-7 green monkey kidney epithelial cells were
transfected with the fusion constructs encoding GFP-Bcl-2,
GFP-Bcl-XL, and GFP-Bax.
Fig. 1.
Distribution of
GFP-fusion proteins expressed in living Cos-7 cells,
before and after STS treatment. At 48 h after transfection, cells were examined by
confocal microscopy. Each
field was visualized by laser
fluorescence to detect GFP
(a, c, e, g, i, k, m, and o), and
by DIC to illustrate cell morphology (b, d, f, h, j, l, n, and
p). Native GFP (a and b) distributes throughout untreated cells, that display a healthy
morphology. In contrast,
GFP-Bcl-2 (e and f) displays
a punctate distribution, mostly
perinuclear. GFP-Bcl-XL (i
and j) appears to distribute in
a mainly punctate pattern
similar to GFP-Bcl-2, but also
has a faint diffuse fluorescence that extends throughout cells. GFP-Bax (m and n)
distributes freely throughout
cells, in a pattern indistinguishable from that of GFP
alone. After 6 h of treatment with 1 µM STS, Cos-7
cells assume a morphology
indicative of apoptosis, including loss of cell volume, retraction of processes, and
blebbing of cell membrane.
Despite these changes, native
GFP (c and d) still distributes throughout the cell. GFP-Bcl-2 (g and h) retains a punctate distribution after STS treatment. GFP-Bcl-XL
(k and l) also does not appear to change distribution with STS treatment, remaining mainly punctate with a background of diffuse fluorescence. However, GFP-Bax (o and p) undergoes a striking change in distribution, becoming entirely punctate after STS treatment.
Bar, 20 µm.
[View Larger Version of this Image (113K GIF file)]
Fig. 2.
Distribution of
GFP-fusion proteins expressed in living L929 cells,
before and after STS treatment. At 48 h after transfection, cells were examined by
confocal microscopy. Each
field was visualized by laser
fluorescence to detect GFP
(a, c, e, g, i, k, m, and o), and
by DIC to illustrate cell morphology (b, d, f, h, j, l, n, and
p). Native GFP (a and b) distributes throughout healthy cells. GFP-Bcl-2 (e and f)
displays a punctate perinuclear distribution. GFP-Bcl-XL
(i and j) appears to have both
punctate and diffuse fractions. GFP-Bax (m and n)
distributes freely throughout
cells. After 6 h of treatment
with 1 µM STS, L929 cells
undergo a loss of cell volume
and retraction and blebbing
of extended processes. Native GFP (c and d) is found
throughout the cells after
STS treatment, including
blebs. GFP-Bcl-2 (g and h)
retains a punctate distribution after STS treatment. After
STS treatment, GFP-Bcl-XL
(k and l) shows a largely
punctate distribution with a
background of diffuse fluorescence. As in Cos-7 cells,
GFP-Bax (o and p) undergoes a change in distribution,
becoming punctate after
STS treament. Bar, 30 µm.
[View Larger Version of this Image (96K GIF file)]
Fig. 3.
Expression of truncated and full-length GFP-Bax, GFP-
Bcl-2, and GFP-Bcl-XL fusion proteins in COS cells. COOH-terminal truncated and full-length GFP fusion constructs of Bax
(lanes a and b), Bcl-2 (lanes c and d) and Bcl-XL (lanes e and f)
were transfected into COS cells. The expression of the fusion
products was monitored by Western blotting of the cell lysates
with human Bax 2D2,
human Bcl-2 8C8, and
universal Bcl-XL
2H12 monoclonal antibodies for the detection of Bax, Bcl-2, and
Bcl-XL, respectively. Arrows a are the expressed fusion products.
Arrows b are likely the in-frame NH2-terminal translational variants. Arrows c represent the endogenous simian Bax, Bcl-2, or
Bcl-XL that cross-react with the above specified antibodies.
[View Larger Version of this Image (44K GIF file)]
Fig. 4.
Effect on cell viability of transient expression of wild-type and COOH-terminal truncated forms of Bcl-2 family members, with and without GFP fused at the NH2-terminus. Cells were
either singly transfected with the GFP fusion constructs or cotransfected with GFP and various Bcl-2 family members. At 48 h
after transfection, cells were treated with STS. The number of
GFP-positive cells was counted within a given field at regular intervals, starting at the time of STS addition (~500 GFP-positive
cells at time zero was typical for each experiment). Changes in
cell viability are displayed as number of glowing cells within a
field and expressed as a percentage of time zero value for that
field. (A) Bcl-2 constructs expressed in L929 cells. (n = 4).
pcDNA3 vector control (), Bcl-2 (
), GFP-Bcl-2 (
), Bcl-2
CT (
), GFP-Bcl-2
CT (
), and Bax (
) (B) Bcl-XL constructs expressed in L929 cells. (n = 4). pcDNA3 vector control
(
), Bcl-XL (
), GFP-Bcl-XL (
), Bcl-XL
CT (
), GFP-Bcl-XL
CT (
), and Bax (
) (n = 4). (C) Bax constructs expressed in
Cos-7 cells. (n = 3). pcDNA3 vector control (
), Bax (
), GFP-Bax (
), Bax
CT (
), GFP-Bax
CT (
).
[View Larger Version of this Image (21K GIF file)]
Fig. 5.
GFP-Bcl-2 and the punctate portion of GFP-Bcl-XL
colocalize with mitochondria in Cos-7 cells. Transiently transfected Cos-7 cells were treated with 20 ng/ml Mitotracker Red
CMXRos to stain mitochondria and then examined by laser fluorescence confocal microscopy. Each field was independently visualized with the appropriate wavelength for GFP (a, d, and g) and
for Mitotracker Red CMXRos (b, e, and h) and then the two images were overlaid (c, f, and i). GFP-Bcl-2 localizes primarily to
mitochondria (a-c). A majority GFP-Bcl-XL also localizes to mitochondria, although there is a faint, diffuse background which is
not punctate (d-f). GFP-Bax does not localize to mitochondria in
healthy cells (g-i). Bar, 20 µm.
[View Larger Version of this Image (60K GIF file)]
Fig. 6.
FRAP analysis of GFP, and
GFP-Bax, and GFP-Bcl-2 in healthy and
cells treated with STS. (A) Fluorescence
recovery visualized in individual treated
cells. In a healthy Cos-7 cell expressing GFP-Bax (a-d), fluorescence recovers
rapidly in a photobleached area. After
treatment with STS, a Cos-7 cell with
punctate GFP-Bax (e-h) no longer recovers after photobleaching. In a healthy Cos-7 cell expressing GFP-Bcl-2 (i-l), recovery
does not occur in the photobleached area.
(B) Quantitative FRAP analysis to determine protein mobilities in cells expressing
GFP alone, GFP-Bax, and GFP-Bcl-2.
Fluorescence intensities during recovery
after photobleach are plotted versus time.
Data was collected at 0.46-s intervals during recovery. GFP and GFP-Bax rapidly
recover with a time course consistent with
diffusion. GFP-Bcl-2 shows minimal recovery, indicating that it is largely immobile. All intensity values were normalized to prebleach intensity (I = 100) and to the
intensity immediately after photobleach (I = 0). Bars, 20 µm.
[View Larger Versions of these Images (66 + 16K GIF file)]
Fig. 8.
GFP-Bax colocalizes with mitochondria in Cos-7 cells after STS treatment. Cos-7 cells transiently expressing
GFP-Bax were treated with 1 µM STS to
induce apoptosis and with 20 ng/ml Mitotracker Red CMXRos to stain for mitochondria and then examined after 4 h by
laser fluorescence confocal microscopy.
The field shown was independently visualized by laser fluorescence confocal microscopy at the appropriate wavelength for
GFP (a) and for Mitotracker Red
CMXRos (b), and the two images were
then overlaid (c). Whereas a majority of
the punctate GFP-Bax appears to localize
to mitochondria, there are also areas
where the GFP-Bax is punctate but which
do not label with the mitochondrial dye (c,
arrow). Bar, 20 µm.
[View Larger Version of this Image (26K GIF file)]
Fig. 9.
GFP-Bax redistribution occurs
before cell shrinkage associated with apoptosis. A field containing three living Cos-7
cells expressing GFP-Bax was followed
over time after addition of 1 µM STS. At
each timepoint, the field was visualized by
laser fluorescence to detect GFP-Bax (a-e
and k-o), and by DIC to illustrate cell morphology (f-j and p-t). Time elapsed
after addition of STS is indicated in the
corner of each of the laser fluorescence
panels. Redistribution of the GFP-Bax
was first detectable for the two cells towards the top of the field at 1 h, 24 min after STS addition (c). Changes in cell shape
first became apparent 12-24 min later, as
evidenced by a retraction of cell outlines
(arrows, i and j). Notice that at these timepoints, the lowermost cell has not yet initiated either GFP-Bax redistribution or cell
shrinkage. GFP-Bax redistribution is first
detectable for the lowermost cell at 2 h,
36 min (n). Bar, 20 µm.
[View Larger Version of this Image (153K GIF file)]
Fig. 10.
GFP-Bax redistribution precedes nuclear fragmentation. A field containing three living Cos-7 cells expressing
GFP-Bax was followed over time after addition of 1 µM STS, and 100 ng/ml of the
nuclear stain bis-benzamide (a-e). In each
panel, laser fluorescence confocal microscopy was used at the appropriate wavelength to visualize GFP (green) and bis-benzamide (blue). Time elapsed after STS
addition is indicated in each panel. After
15 h (f) cells show fragmented nuclei associated with apoptosis. Bar, 20 µm.
[View Larger Version of this Image (28K GIF file)]
CT constructs of GFP-Bcl-2 (Fig. 11, a and c), GFP-Bcl-XL
(Fig. 11, e and g), and GFP-Bax (Fig. 11, i and k) all display a diffuse distribution in healthy cells, consistent with
localization throughout the cytosol, when expressed in either Cos-7 or L929 cell lines. Induction of apoptosis causes
no apparent redistribution of any of the
CT constructs in
either cell line (Fig. 11). This indicates that the COOH terminus may play a pivotal role not only in anchoring Bcl-2
and Bcl-XL to organelles in healthy cells, but also in mediating the organelle association of Bax observed in cells undergoing apoptosis.
Fig. 11.
Distribution of
GFP-fusion proteins lacking
COOH-terminal hydrophobic domains before and after
STS treatment. Cos-7 and
L929 cells were transiently
transfected with the appropriate construct, and examined 48 h later by confocal
microscopy. Each field was visualized by laser fluorescence to detect GFP. In both
healthy Cos-7 cells (a) and
L929 cells (c), CT GFP-
Bcl-2 was diffusely distributed throughout the cytosol.
Likewise, in healthy Cos-7 cells (e) and L929 cells (g),
CT GFP-Bcl-XL was diffusely distributed throughout
the cytosol. Finally, in both
healthy Cos-7 cells (i) and
L929 cells (k),
CT GFP-
Bax was diffusely distributed
throughout the cytosol. Cells
expressing truncated GFP-fusion proteins were treated
with 1 µM STS, and examined after 6 h. In both Cos-7
cells (b, f, and j) and L929
cells (d, h, and l), none of the
truncated proteins redistributed after STS treatment.
Bar, 20 µm.
[View Larger Version of this Image (59K GIF file)]
CT would be expected to have
diminished potential to enhance apoptosis relative to wild-type Bax. The bioactivity of
CT constructs of Bax, Bcl-2,
and Bcl-XL was compared to that of the full-length genes
in a cell viability assay both with and without GFP fused to
the NH2 terminus of each protein upon induction of apoptosis with STS. As illustrated in Fig. 4, A and B, for both
Bcl-2 and Bcl-XL, deletion of the COOH-terminal hydrophobic domain resulted in a decrease in the apoptosis protection in L929 cells. For Bax, the deletion of the COOH-terminal domain also resulted in a sharp decrease in the
ability of both wild-type and GFP-linked protein to accelerate apoptosis in both Cos-7 (Fig. 4 C) and L929 cells
(data not shown). These results indicate that the COOH-terminal hydrophobic domain is important for Bax function, linking the ability of Bax to redistribute to organelles
during apoptosis with its death-accelerating activity.
Discussion
Footnotes
Address all correspondence to Richard J. Youle, Bldg 10, Room 5D-37,
National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 496-6628. Fax: (301) 402-0380. E-mail: youle{at}helix.nih.gov
Abbreviations used in this paper
CT, COOH-terminal truncation;
DIC, differential interference contrast;
GFP, green fluorescent protein;
STS, staurosporine.
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