Am J Physiol Cell Physiol 300: C447–C455, 2011.
First published December 15, 2010; doi:10.1152/ajpcell.00402.2010.
Fragmented mitochondria are sensitized to Bax insertion and activation
during apoptosis
Craig Brooks,1 Sung-Gyu Cho,1 Cong-Yi Wang,2 Tianxin Yang,3 and Zheng Dong1,4
1
Department of Cellular Biology and Anatomy, 2Center for Biotechnology and Genomic Medicine, Medical College of
Georgia, Augusta, Georgia; 3Department of Internal Medicine, University of Utah, Salt Lake City, Utah; and 4Renal
Research Laboratory, Charlie Norwood Veterans Affairs Medical Center, Augusta, Georgia
Submitted 28 September 2010; accepted in final form 13 December 2010
mitochondrial dynamics; mitochondria outer membrane permeabilization; cytochrome c
CELLULAR STRESS,
if severe enough, frequently triggers the
intrinsic pathway of apoptosis, which is characterized by mitochondrial outer membrane permeabilization (MOMP) (15,
28, 30). MOMP leads to the release from the intermembrane
space of several apoptogenic factors or proteins, including
cytochrome c (Cyt c) and apoptosis-inducing factor (AIF).
Whereas Cyt c is well documented for its ability to initiate
the formation of apoptosome to result in caspase activation,
AIF is suggested to induce caspase-independent apoptosis
(15, 28, 30).
During apoptosis, MOMP is mainly mediated and regulated
by Bcl-2 family proteins, which are characterized by the
presence of Bcl-2 homology (BH) domains (1, 9, 11, 33).
Address for reprint requests and other correspondence: Z. Dong, Dept. of
Cellular Biology and Anatomy, Medical College of Georgia, 1459 Laney
Walker Blvd., Augusta, GA 30912 (e-mail: [email protected]).
http://www.ajpcell.org
Depending on their functions in apoptosis, Bcl-2 family proteins are divided into pro- and anti-apoptotic members. Structurally, the anti-apoptotic members, such as Bcl-2 and Bcl-XL,
contains four BH domains (BH1-BH4), whereas the pro-apoptotic members either have three BH domains or only one BH
domain, the BH-3 (1, 9, 11, 33). It is now broadly appreciated
that Bax and Bak, two multi-BH domain pro-apoptotic members, are the molecular gateway or mediators of MOMP, which
are inhibited by the anti-apoptotic Bcl-2/Bcl-XL under normal
conditions or activated by BH3-only proteins during apoptosis.
Whereas both Bax and Bak contribute to the development of
MOMP, they are distinctly regulated. Especially, Bax resides
in cytosol in normal healthy cells, whereas Bak is in mitochondria. As a result, the regulation of Bax during apoptosis is
apparently more complex, involving translocation to mitochondria, insertion into the outer membrane, and activation and
formation of oligomers. Despite years of investigation, how
each of the Bax activation events is regulated, culminating in
mitochondrial permeabilization and the release of apoptogenic
factors, remains largely unclear (1, 9, 11, 33).
Recent work has suggested a role for alterations of mitochondrial dynamics in the development of MOMP during
apoptosis (3, 6, 7, 25, 26). Mitochondria are a class of dynamic
organelles, constantly undergoing fission and fusion (7, 21).
Interestingly, mitochondrial fission and fusion are governed by
distinct proteins. Whereas fission depends on Fis-1 and Drp1,
fusion requires a coordinated action of Mitofusins and OPA-1
(7, 21). Under normal conditions, mitochondrial fusion prevails and as a result, the organelles appear long and filamentous. During cell stress, however, the dynamic balance is
shifted to fission, leading to mitochondrial fragmentation (5,
13, 19, 27). Importantly, emerging evidence has suggested that
mitochondrial fragmentation contributes MOMP and consequent release of apoptogenic factors during apoptosis (3, 6, 7,
25, 26). Despite these findings, it is unclear as to how mitochondrial fragmentation, a seemingly morphological change,
can affect MOMP, the formation of porous defects in mitochondrial outer membrane.
In this study, we have further confirmed that prevention of
mitochondrial fragmentation, either by blocking fission or
by promoting fusion, can suppress MOMP and apoptosis
following cellular stress. In addition, we have shown that cells
with fragmented mitochondria are more sensitive to MOMP
and apoptosis. Importantly, we have demonstrated that fragmented mitochondria are sensitized to Bax insertion and activation, suggesting a mechanism for the involvement of mitochondrial fragmentation in the development of MOMP during
apoptosis.
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Brooks C, Cho S, Wang C, Yang T, Dong Z. Fragmented
mitochondria are sensitized to Bax insertion and activation during
apoptosis. Am J Physiol Cell Physiol 300: C447–C455, 2011. First
published December 15, 2010; doi:10.1152/ajpcell.00402.2010.—Recent studies have shown mitochondrial fragmentation during cell
stress and have suggested a role for the morphological change in
mitochondrial injury and ensuing apoptosis. However, the underlying
mechanism remains elusive. Here we demonstrate that mitochondrial
fragmentation facilitates Bax insertion and activation in mitochondria,
resulting in the release of apoptogenic factors. In HeLa cells, overexpression of mitofusins attenuated mitochondrial fragmentation during cisplatin- and azide-induced cell injury, which was accompanied
by less apoptosis and less cytochrome c release from mitochondria.
Similar effects were shown by inhibiting the mitochondrial fission
protein Drp1 with a dominant negative mutant (dn-Drp1). Mitofusins
and dn-Drp1 did not seem to significantly affect Bax translocation/
accumulation to mitochondria; however, they blocked Bax insertion
and activation in mitochondrial membrane. Consistently, in rat kidney
proximal tubular cells, small interfering RNA knockdown of Drp1
prevented mitochondrial fragmentation during azide-induced ATP
depletion, which was accompanied by less Bax activation, insertion,
and oligomerization in mitochondria. These cells released less cytochrome c and AIF from mitochondria and showed significantly lower
apoptosis. Finally, mitofusin-null mouse embryonic fibroblasts (MEF)
had fragmented mitochondria. These MEFs were more sensitive to
cisplatin-induced Bax activation, release of cytochrome c, and apoptosis. Together, this study provides further support for a role of
mitochondrial fragmentation in mitochondrial injury and apoptosis.
Mechanistically, mitochondrial fragmentation may sensitize the cells
to Bax insertion and activation in mitochondria, facilitating the release
of apoptogenic factors and consequent apoptosis.
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MATERIALS AND METHODS
Cells and reagents. Mfn1⫺/⫺, Mfn2⫺/⫺, and control wild-type (wt)
mouse embryonic fibroblasts (MEF) were kindly provided by Dr.
David Chan at California Institute of Technology (Pasadena, CA).
The cells were characterized previously (8). The rat kidney proximal
tubular cell line (RPTC) was originally obtained from Dr. Ulrich
Hopfer at Case Western Reserve University (Cleveland, OH). The R3
and R24 cell clones were generated by stable transfection of RPTC
cells with short hairpin RNAs (shRNAs) targeting Drp1 as described
in our recent work (4). HeLa cells were purchased from American
Type Culture Collection (ATCC, Manassas, VA). Mfn1 and Mfn2
plasmids were obtained from Dr. David Chan, and dn-Drp1 was from
Dr. Alexander van der Bliek at the University of California School of
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condensation, formation of apoptotic bodies, and condensation and
fragmentation of the nucleus. About 200 cells were examined in each
dish to determine the percentage of apoptotic cells.
Analysis of the release of Cyt c and AIF. Cyt c and AIF normally
reside in mitochondria in the intermembrane space, and upon apoptotic stimulation, they are released from mitochondria. To determine the
release of these proteins during apoptosis, cells were fractionated by
using digitonin at low concentrations (4, 12, 24, 29). Briefly, cells
were permeabilized with 0.05% (wt/vol) digitonin in an isotonic
sucrose buffer for 2– 4 min. The cytosol released by digitonin from the
same number cells was collected from different treatment groups for
immunoblot analysis using specific antibodies to Cyt c or AIF.
Immunofluorescence of Bax. Indirect immunofluorescence of Bax
was conducted as described in our previous studies (4, 12, 24, 29).
Briefly, cells grown on glass coverslips were fixed with a modified
Zamboni’s fixative containing picric acid and 4% paraformaldehyde.
The fixed cells were incubated with a blocking buffer containing 2%
normal goat serum and then exposed to the primary anti-Bax antibody.
Finally, the cells were incubated with FITC-labeled goat anti-mouse
secondary antibody. The signals were examined by fluorescence
microscopy.
Immunoprecipitation analysis of active Bax. Bax activation involves the conformational changes to expose the NH2-terminus of the
protein. As a result, antibodies specific to the NH2-terminal sequence
of Bax only bind and react with active Bax. To detect Bax activation,
in this study we used the anti-Bax NT antibody from Upstate Biotechnology that was generated against the Bax NH2-terminal sequence
for immunoprecipitation to pull down active Bax. Briefly, cell lysate
was collected in the immunoprecipitation buffer containing CHAPS.
The lysate of 500 ␮g protein was subjected to immunoprecipitation by
incubation with 1 ␮g anti-Bax NT and protein A/G agarose beads.
After centrifugation and washes, the precipitated proteins were collected and examined by immunoblot analysis.
Analysis of Bax insertion by alkaline treatment. Exposure to an
alkaline buffer can strip off the loosely attached proteins, but not the
inserted proteins, from mitochondria. Alkaline exposure was conducted as described in our previous work (32). Briefly, cells were
permeabilized with 0.05% digitonin to release cytosolic fraction. The
membrane-bound organellar fraction containing mitochondria was
collected, washed with PBS, and then incubated on ice in 0.1 M
Na2CO3 at pH 11.5 for 30 min. After 1 h of centrifugation at 100,000
g, the pellet was collected for immunoblot analysis to reveal the
inserted Bax that was resistant to alkaline stripping.
Analysis of Bax oligomerization. Bax oligomerization was analyzed
following chemical cross-linking as described in our previous work
(16). Briefly, cells were permeabilized with 0.05% digitonin to release
cytosolic fraction. After centrifugation, the membrane-bound organellar fraction containing mitochondria was collected and incubated
with 1 mM dithiobis[succinimidyl propionate] (DSP from Pierce,
Rockford, IL) for 30 min of cross-linking at room temperature. The
cross-linked samples were subjected to immunoblot analysis of Bax
under nonreducing conditions.
Fig. 1. Expression of Mfn1, Mfn2, and dn-Drp1 inhibits mitochondrial fragmentation, cytochrome c (Cyt c) release, and apoptosis. A: representative
mitochondrial morphology. HeLa cells were cotransfected with Mito-Red and with one of the following plasmids: dn-Drp1, Bcl-2, Mfn1, Mfn2, or empty vector.
After overnight transfection, the cells were untreated or treated with 10 mM azide for 3 h. Mitochondrial morphology was examined and recorded by fluorescence
microscopy. Insets: boxed area in higher magnification. B: mitochondrial fragmentation during cisplatin treatment. HeLa cells were cotransfected with MitoRed
and Mfn1, Mfn2, dn-Drp1, or empty vector. The cells were then treated with 20 ␮M cisplatin for 16 h to evaluate mitochondrial morphology by fluorescence
microscopy. The cells containing fragmented mitochondria and those with filamentous mitochondria were counted to calculate the percentage of cells with
mitochondrial fragmentation. C: apoptosis during cisplatin treatment. HeLa cells were transfected as described in A and treated with cisplatin for 24 h to examine
apoptosis by morphological criteria. The percentage of apoptosis was determined by counting the cells with typical apoptotic morphology. D: Cyt c release during
cisplatin treatment. HeLa cells were transfected as described in A and treated with cisplatin for 24 h. The cells were premeabilized with low concentration
digitonin to collect the cytosolic fraction to analyze the released Cyt c by immunoblotting. E: Cyt c release during azide treatment. HeLa cells were transfected
as described in A and then subjected to 3 h of ATP depletion with 10 mM azide treatment in glucose-free buffer. The cells were premeabilized with low
concentration digitonin to collect the cytosolic fraction to analyze the released Cyt c by immunoblotting. Data in are B and C are expressed as means ⫾ SD
(n ⫽ 3); *significantly different from the cisplatin-treated empty vector-transfected group.
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Medicine at Los Angeles. pDsRed2-Mito (MitoRed) and pAcGFP1Mito (MitoGreen) was purchased from BD Clontech (Palo Alto, CA).
Antibodies were from the following sources: mouse monoclonal
anti-Cyt c from BD Pharmingen (San Diego, CA); mouse monoclonal
anti-Bax (1D1, specific for rat Bax) from MeoMarkers (Fremont, CA),
rabbit polyclonal anti-Bax (for human, mouse Bax) from Santa Cruz
Biotechnology (Santa Cruz, CA); rabbit polyclonal anti-Bax (NT,
specific for active Bax) and anti-Bak from Upstate (Lake Placid, NY);
and secondary antibodies from Jackson ImmunoResearch (West
Grove, PA). Other reagents and chemicals including cisplatin and
azide were purchased from Sigma (St. Louis, MO).
Cell injury models. Cells were treated with cisplatin or azide to
induce cellular stress and injury by protocols modified from our
previous studies (4, 5, 12, 17, 22, 29). Cisplatin is a chemotherapy
drug that induces apoptosis in tumors and normal tissues by multiple
mechanisms including DNA damage. In this study, cells were incubated with indicated concentrations of cisplatin in culture medium.
Azide blocks cellular respiration at complex IV. In this study, cells
were incubated with 10 mM azide in glucose-free medium, a treatment that leads to ATP depletion and consequent mitochondrial
leakage of apoptogenic factors such as Cyt c. When the azide-treated
cells are returned to glucose-containing medium, the cells develop
apoptotic morphology.
Plasmid transfection. Cells were plated at 40 – 60% confluence for
transfection using Lipofectamine LTX with Plus Reagent (Invitrogen). Usually, 1 ␮g plasmid DNA was transfected to cells in each
35-mm dish. To reveal mitochondrial morphology, 0.5 ␮g pDsRed2mito plasmid or pAcGFP1-Mito was transfected alone or 0.1 ␮g
cotransfected with other plasmids. The transfected cells were subjected to indicated treatments and examined after overnight growth.
Analysis of mitochondrial morphology. Mitochondrial morphology
was examined as described in our recent studies (4, 5, 10). Briefly,
cells were transfected with pDsRed2-Mito or pAcGFP1-Mito, which
led to the expression of MitoRed or MitoGreen mitochondrial-targeted fluorescent proteins, in mitochondria to label the organelles. The
transfected cells were then subjected to control or experimental
treatments to evaluate mitochondrial morphology by fluorescence
microscopy. Filamentous mitochondria showed a long thread-like
tubular structure, whereas fragmented mitochondria were punctate
and sometimes rounded. For quantification, the cells with different
mitochondrial morphologies were counted to determine the percentage of cells with fragmented mitochondria. Most of the cells had
either fragmented or filamentous mitochondria, whereas a small percentage (⬍10%) of cells contained both fragmented and filamentous
mitochondria. If a cell happened to have mitochondria with mixed
morphologies, we classified the mitochondrial morphology according
to the majority (⬎70%) of the mitochondria.
Evaluation of apoptosis. Apoptosis was examined morphologically
as described in our previous work (4, 5, 10, 12, 17, 22, 29). Briefly,
after various treatments, cells were stained with Hoechst 33342 and
examined by phase-contrast and fluorescence microscopy. Apoptotic
cells were identified by characteristic morphology including cellular
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Immunoblot analysis. Protein concentration of cell lysate was
determined by using the bicinchoninic acid reagent (Pierce, Rockford,
IL). Immunoblot analysis was performed by a standard protocol.
Briefly, equal amount of proteins or protein samples collected from
the same number of cells were resolved by reducing SDS-gel electrophoresis and electroblotted onto PVDF membranes. The blots were
incubated sequentially with a blocking solution, a specific primary
antibody, and a horseradish-peroxidase-conjugated secondary antibody. Finally, antigens on the blots were revealed using the enhanced
chemiluminescence kit (Pierce).
Statistics. Quantitative data were analyzed by Student’s test and
expressed as means ⫾ SD. Statistical differences between the means
were determined using analysis of variance (ANOVA) followed by
Tukey’s post test. P ⬍ 0.05 was considered to reflect significant
differences. Qualitative data including cell images and immunoblots
were representatives of at least three experiments.
Expression of Mfn1, Mfn2, or dn-Drp1 inhibits mitochondrial fragmentation, Cyt c release, and apoptosis. Our previous
work showed that HeLa cells had long and filamentous mitochondria, which became fragmented during cell stress. Moreover, blockade of mitochondria fragmentation by expressing a
dominant negative mutant of Drp1 could suppress mitochondrial outer membrane leakage, the release of apoptogenic
factors (e.g., Cyt c) and attenuate subsequent apoptosis (4, 5).
While those results suggest a critical role for mitochondrial
fragmentation in apoptosis, it can be argued that Drp1 per se
(and not mitochondrial fragmentation) is the key. To address
this question, we employed a different approach to prevent
mitochondrial fragmentation, i.e., by expressing Mitofusins
Mfn1 and Mfn2. Two cell injury models, involving the use of
the mitochondrial respiration inhibitor azide and the chemotherapy drug cisplatin, respectively, were examined. As shown
in Fig. 1A, control HeLa cells contained long filamentous
mitochondria, which became small discrete fragments during
azide treatment. Transfection with Mfn1 or 2 prevented azideinduced mitochondrial fragmentation. Consistent with our previous study (5), mitochondrial fragmentation was also attenuated by dn-Drp1 and Bcl-2 (Fig. 1A). In the cisplatin injury
model, we quantified the effects by counting the cells with
fragmented mitochondria (Fig. 1B). In empty vector-transfected cells, cisplatin induced mitochondrial fragmentation in
45% cells, which was suppressed to 20 –25% by Mfn1, Mfn2,
and dn-Drp1. Importantly, transfection with Mfn1, Mfn2, or
dn-Drp1 significantly suppressed cisplatin-induced apoptosis
(Fig. 1C). At the mitochondrial level, we further showed that
Mfn1 and Mfn2 as well as dn-Drp1 and Bcl-2 could block the
release of Cyt c (Cyt c) during cisplatin and azide treatments
(Fig. 1, D and E). Together, these results suggest that maintenance of mitochondrial morphological dynamics during cell
stress by either blocking fission (via dn-Drp1) or enhancing
fusion (via Mfn1, Mfn2) can suppress mitochondrial injury and
consequent apoptosis.
Mfn1, Mfn2, and dn-Drp1 do not block Bax translocation to
mitochondria during cell stress. Bax and Bak are considered
the “gateway” to mitochondrial outer membrane permeabilization and the release of apoptogenic factors during apoptosis (1,
9, 11, 31, 33). Whereas Bak is constitutively localized in
mitochondria, Bax normally resides in the cytosol and translocates to mitochondria upon apoptotic stimulation. At mitochondria, Bax is further activated to insert into the outer
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Fig. 2. DN-Drp1, Mfn1, and Mfn2 do not block Bax translocation to mitochondria during azide treatment. A: Bax translocation analyzed by immunofluorescence staining. HeLa cells were cotransfected with MitoRed and one
indicated plasmid (empty vector, dn-Drp-1, Bcl-2, Mfn1, or Mfn2). After azide
treatment, the cells were fixed for Bax immunofluorescence staining (labeled
by green FITC) and examined to count the cells with Bax translocation to
mitochondria. Data are expressed as means ⫾ SD (n ⫽ 4); *P ⬍ 0.01 vs.
control; #P ⬍ 0.01 vs. azide treated vector-transfected group. B: Myc-Mfn1
and Myc-Mfn2 expression after transfection. Whole cell lysate was collected
for immunoblot analysis using specific antibodies to Mfn1, Mfn2, Drp1, and
␤-actin.
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RESULTS
membrane and form oligomers, which may directly release the
apoptogenic factors or indirectly by destabilizing the membrane lipids (1, 9, 11, 33). Thus to understand how mitochondrial fragmentation affects mitochondrial injury, we examined
the effects of Mfn1, Mfn2, and dn-Drp1 expression on Bax
accumulation to mitochondria by immunofluorescence staining
(Fig. 2A). For this purpose, cells were cotransfected with
MitoRed and one indicated plasmid (empty vector, dn-Drp-1,
Bcl-2, Mfn1 or Mfn2). After azide treatment, the cells were
subjected to Bax immunofluorescence staining (labeled by
green FITC) and examined to count the cells with Bax staining
in mitochondria. As shown in Fig. 2B, except Bcl-2, none of
the transfected genes prevented Bax accumulation to mitochondria during azide treatment. By immunoblot analysis, we
confirmed the expression of Myc-Mfn1 and -Mfn2 after transfection, which did not affect the expression of Drp1 (Fig. 2B).
Mfn1, Mfn2, and dn-Drp1 suppress Bax activation and
insertion in mitochondria membrane. We then examined
whether Mfn1, Mfn2, and dn-Drp1 could inhibit Bax activation
and insertion into mitochondria. Bax activation was detected
by immunoprecipitation using the NT anti-Bax antibody,
which was generated by Bax NH2-terminal sequence and
specifically recognizes active Bax. As shown in Fig. 3A, active
Bax was not detected in untreated cells (lane 1), whereas
abundant active Bax was pulled down from azide-treated cell
lysate (lane 2). Notably, much less active Bax was precipitated
from the cells tranfected with Mfn 1, Mfn2, or dn-Drp1 (lanes
3, 5, and 6). As a positive control, Bax activation was confirmed to be suppressed by Bcl-2 (lane 4). We further analyzed
Bax insertion into mitochondrial membrane. To this end, the
membrane fraction containing mitochondria was isolated and
exposed to an alkaline buffer, which can strip off uninserted
proteins (14, 32). As shown in Fig. 3B, after alkaline stripping
MITOCHONDRIA FRAGMENTATION IN BAX ACTIVATION
very little Bax remained in mitochondria in untreated control
cells, but still significant amounts of Bax remained in the
mitochondria of azide-treated cells (lane 2), indicating Bax
insertion into mitochondria membrane. Azide-induced Bax
insertion was completely blocked by dn-Drp1 and Bcl-2 and
partially attenuated by Mfn1 and Mfn2 expression. Together
the results suggest that mitochondrial fragmentation facilitates
Bax insertion to and activation in mitochondrial membrane.
Knockdown of Drp1 inhibits Bax activation, insertion, and
oligomerization in mitochondria. To further confirm the role of
mitochondrial fragmentation in Bax regulation in other cell
types, we examined RPTC, a kidney epithelial (proximal tubular) cell line. RPTC was used in our recent work to reveal a
pathological role for alterations of mitochondrial dynamics in
acute kidney injury (4). In that study, we generated two stable
cell lines (R3, R24) with Drp1 knockdown via shRNA. We
therefore examined azide-induced Bax activation in these cells.
We confirmed that the shDrp1 cells were resistant to azideinduced mitochondrial fragmentation [Fig. 4A, quantitative
data published previously (4)]. These cells were also markedly
resistant to apoptogenic factor release during azide treatment,
including Cyt c (Ref. 4) and AIF (Fig. 4B). Importantly, when
compared with the parental RPTC, both R3 and R24 cells
showed obviously lower Bax activation and mitochondrial
insertion during azide treatment (Fig. 4C). We further analyzed
the formation of Bax oligomers after chemical cross-linking of
the cells. As shown in Fig. 4D, azide treatment induced the
formation of Bax dimmer and trimer in RPTC, which was
almost completely blocked in R3 cells and partially inhibited in
Fig. 4. Knockdown of Drp1 inhibits Bax activation, insertion and oligomerization in mitochondria. R3 and R24 cell clones were generated by stable transfection
of Drp-1 short hairpin RNA (shRNA) into rat proximal tubular cells (RPTC). Knockdown of Drp1 in R3 and R24 cells was shown in our recent work.
A: representative images of mitochondrial morphology. RPTC and R24 cells were transfected with Mito-Green and then left untreated (control) or treated with
10 mM azide for 3 h to record mitochondrial morphology by fluorescence microscopy. B: apoptosis-inducing factor (AIF) release. Cells were untreated or treated
with 10 mM azide for 3 h. The cells were then permeabilized with low concentration digitonin to collect cytosolic fraction for immunoblot analysis of released
AIF. C: Bax activation and insertion in mitochondria. RPTC, R3, and R24 cells were untreated or treated with 10 mM azide for 3 h. To analyze Bax activation,
cell lysate was collected for immunoprecipitation using an antibody recognizing active Bax, followed by immunoblot analysis of Bax. To analyze Bax insertion,
membrane fraction containing mitochondria was isolated and subjected to alkaline (pH 11.5) incubation as described in MATERIALS AND METHODS. The fraction
was then collected by centrifugation for analysis of remaining Bax by immunoblot analysis. Bak was also analyzed to verify protein loading. D: Bax
oligomerization. RPTC, R3, and R24 cells were untreated or treated with 10 mM azide for 3 h. The cells were then cross-linked with 1 mM DSP and further
permeabilized with digitonin to collect the membrane fraction containing mitochondria. The membrane fraction was finally subjected to nonreducing gel
electrophoresis and immunoblot analysis of Bax.
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Fig. 3. DN-Drp1, Mfn1, and Mfn2 suppress Bax activation and insertion in
mitochondria membrane. HeLa cells were transfected with Mfn1, Mfn2,
dn-Drp1, Bcl-2, or control empty vector. The cells were then subjected to 3 h
of 10 mM azide treatment in glucose-free buffer. A: active Bax. Cells lysate
was collected for immunoprecipitation with an antibody that was specific to
active Bax. The precipitate was finally analyzed by immunoblotting of Bax.
B: Bax insertion. Membrane fraction containing mitochondria was collected
from the cells and incubated for 30 min with an alkaline (pH 11.5) solution.
The mitochondrial fraction was then collected by centrifugation for analysis of
remaining Bax by immunoblot analysis. Bak was also analyzed to verify that
inserted proteins were not stripped off from mitochondrial membrane by the
alkali incubation.
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R24 cells. Azide-induced apoptosis was attenuated in R3 and
R24 cells (data not shown, published in Ref. 4). The results
further indicate that mitochondrial fragmentation contributes to
Bax activation and insertion in mitochondria, leading to the
release of apoptogenic factors and apoptosis.
Mfn1 or Mfn2-null cells have fragmented mitochondria. By
promoting fusion (Mfn1, Mfn2) or inhibiting fission (dn-Drp1,
siDrp1), the experiments described above showed that keeping
mitochondria in a long filamentous shape can suppress Bax
activation and insertion in mitochondrial membrane, resulting
in suppression of mitochondrial leakage and attenuation of
apoptosis. To further validate this conclusion, we went on to
examine cells that started with fragmented mitochondria. MEF
originated from Mfn1- or Mfn2-null mice have significantly
higher mitochondrial fragmentation than wt MEF (8). We
confirmed mitochondrial fragmentation in these cells (Fig. 5A).
Fig. 6. Mfn1 or MF2-null cells are more sensitive to apoptosis. wt, Mfn1-null, and Mfn2-null MEFs were incubated with 20 ␮M cisplatin for 24 h to record cell
morphology. Apoptotic cells were identified by typical morphology including cellular condensation and fragmentation. A: representative cell morphology. Note:
many Mfn1 or Mfn2-null cells had undergone apoptosis and detached form the dish. Arrows: representative apoptotic cells. B: percentage of apoptosis. Data are
means ⫾ SD (n ⫽ 3); *significantly different from wt cells treated with cisplatin.
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Fig. 5. Mfn1 or MFn2-null cells have fragmented mitochondria. A: representative mitochondrial morphology. Wild-type (wt), Mfn1null, and Mfn2-null mouse embryonic fibroblast (MEF) cells were transfected with
MitoRed to record mitochondrial morphology
by fluorescence microscopy. B: quantification
of cells with fragmented mitochondria. Cells
were transfected as those cells in A and examined by fluorescence microscopy to determine
the percentage of cells with fragmented mitochondria. C: mitochondrial fragmentation during cisplatin treatment. wt, Mfn1-null, and
Mfn2-null MEF cells were transfected with
MitoRed and then treated with 20 ␮M cisplatin
for 16 h. The cells were examined by fluorescence microscopy to determine the percentage
of cells with fragmented mitochondria. Data
are means ⫾ SD (n ⫽ 3); *significantly different
wt cells (B) or cisplatin-treated wt group (C).
MITOCHONDRIA FRAGMENTATION IN BAX ACTIVATION
DISCUSSION
Changes of mitochondrial dynamics, resulting in fragmentation of the organelles, have been documented during cell
stress in a variety of apoptotic models. Emerging evidence has
further suggested an important role for mitochondrial fragmentation in mitochondrial injury during apoptosis (3, 7, 25, 26).
Importantly, the pathological role of mitochondrial fragmentation has been demonstrated not only by in vitro cell culture
studies but also by in vivo animal model examination. Our
recent work (4) has revealed fragmented mitochondria in renal
tubular cells during renal ischemia-reperfusion injury and cisplatin-induced nephrotoxicity in mice. Notably, pharmacological inhibition of mitochondrial fragmentation can partially, but
significantly, protect the kidneys under the injury conditions.
Despite these observations, whether and to what extent mitochondrial fragmentation contributes to apoptosis has been
questioned by a few other studies (2, 23). Especially, it has
been questioned whether mitochondrial fragmentation is a
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Fig. 7. Mfn1 or Mfn2-null cells are more sensitive to Cyt c release, Bax
activation, and insertion. wt, Mfn1-null, and Mfn2-null MEFs were incubated
with 20 ␮M cisplatin for 24 h. A: Cyt c release. Cells were permeabilized with
low concentration digitonin to collect cytosolic fraction for immunoblot
analysis to detect Cyt c that had been released into cytosol during cisplatin
treatment. B: Bax activation. Cells were lysed with the CHAPS buffer. The
lysate was subjected to immunoprecipitation using the antibody specific for
active Bax. The resultant immunoprecipitates were analyzed for Bax by
immunoblot analysis. C: Bax insertion. Cells were permeabilized with digitonin to release cytosol and collect the membrane fraction with mitochondria,
which was subjected to alkaline incubation as described in MATERIALS AND
METHODS. After alkaline treatment, Bax remaining in the membrane fraction
was analyzed by immunoblot analysis.
cause or just a consequence of MOMP or mitochondrial outer
membrane permeabilization. In addition, as blocking mitochondrial fragmentation in previous studies was mainly
achieved by expressing dominant negative Drp1, it has been
speculated that maybe it is Drp1 (and not mitochondrial fragmentation) that somehow contributes to MOMP and apoptosis.
Finally, it remains largely unknown how changes in mitochondrial dynamics affect mitochondrial injury or MOMP during
apoptosis.
The results from the current study have provided information to address the critical questions listed above. First, we
have further demonstrated compelling evidence to support a
role for mitochondrial fragmentation in the development of
MOMP and apoptosis. We show that prevention of mitochondrial fragmentation, either by blocking fission or by enhancing
fusion, can suppress MOMP as indicated by lowered release of
Cyt c and AIF (Figs. 1, C and D, and 4B). Moreover, upon
apoptotic induction, MOMP is exacerbated in cells with fragmented mitochondria (Fig. 7A). Of note, these experiments
were conducted in several apoptotic models involving different
types of apoptotic treatment (azide, cisplatin) and cells (HeLa,
RPTC, MEF). Second, a main approach of this study is ma-
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Quantification by cell counting indicated that ⬃60% Mfn1⫺/⫺
and ⬃80% Mfn2⫺/⫺ cells had fragmented mitochondria,
whereas less than 10% wt MEF had (Fig. 5B). Interestingly, in
both wt and Mfn⫺/⫺ cells, cisplatin stress could increase
mitochondrial fragmentation. After the treatment, mitochondrial fragmentation reached the maximal level of 80 –90% in
Mfn1 or Mfn2-null cells (Fig. 5C).
Mfn1 or Mfn2-null cells are more sensitive to apoptosis. The
Mfn-null cells with high background mitochondrial fragmentation provided us a model for further investigation of the
effect of mitochondrial fragmentation on cellular sensitivity to
mitochondrial injury and apoptosis. Under control conditions,
wt and Mfn-null cells did not show obvious apoptosis, despite
mitochondrial fragmentation in the latter. Our initial experiments examined cisplatin-induced apoptosis in these cells. The
wt MEFs were not very sensitive to cisplatin treatment and, as
a result, only induced sparse apoptosis by 20 ␮M cisplatin
(Fig. 6A: left). In sharp contrast, apoptosis was wide spread in
Mfn1-null and Mfn2-null MEFs following the same cisplatin
incubation (Fig. 6A: middle and right). By counting the cells
with apoptotic morphology, it was estimated that cisplatin
induced about 30% and 50% apoptosis in Mfn1-null and
Mfn2-null cells, whereas only 10% was induced in wt MEFs
(Fig. 6B).
Mfn1 or Mfn2-null cells are more sensitive to Cyt c release,
Bax activation, and insertion. The higher sensitivity of Mfnnull cells to cisplatin-induced apoptosis (shown above in Fig.
6) promoted us to analyze the mitochondrial events of apoptosis in these cells. Under control conditions, Mfn-null cells as
wild-type cells did not show Bax activation or Cyt c release
(not shown). During cisplatin treatment both Mfn1-null and
Mfn2-null cells released significantly more Cyt c from mitochondria than wild-type cells (Fig. 7A: lanes 3, 4 vs. lane 2).
Notably, cisplatin induced remarkably higher Bax activation in
Mfn-null cells than wild-type cells (Fig. 7B: lanes 3, 4 vs. lane
2). In addition, more Bax was inserted into mitochondria in
Mfn-null cells (Fig. 7C: lanes 3, 4 vs. lane 2). The results
suggest that cells with fragmented mitochondria are sensitized
to Bax activation and insertion, resulting in mitochondrial
leakage of apoptogenic factor and activation of the apoptotic
cascade.
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It is currently unclear why fragmented mitochondria are
sensitized to Bax insertion and activation. We speculate there
are at least two possibilities. First, the biophysical property of
the membrane of filamentous mitochondria may be different
from that of fragmented mitochondria. In this regard, apparently there are differences in the membrane curvatures in
filamentous and fragmented mitochondria. It would be interesting to determine whether membranes with different curvatures show different sensitivity to Bax insertion. Second, when
mitochondria change their fission-fusion dynamics and undergo fragmentation, there may be significant changes in the
biochemical property of the membrane. In this regard, both
proteins and lipids may have changes that affect the docking
and insertion of Bax into the membrane. Future research
should investigate these possibilities to advance the understanding of mitochondrial dynamics and injury during cell
stress.
ACKNOWLEDGMENTS
We thank Dr. David Chan at California Institute of Technology for kindly
providing the Mfn1 and Mfn2 plasmids and the Mfn1⫺/⫺, Mfn2⫺/⫺, and wt
MEFs. We also thank Dr. Alexander van der Bliek at the University of
California School of Medicine at Los Angeles for the dominant negative Drp1
plasmid.
Present address of C. Brooks: Renal Division, Dept. of Medicine, Brigham
and Women’s Hospital, Harvard Medical School, Boston, MA 02115.
GRANTS
Z. Dong is a Research Career Scientist of Department of Veterans Affairs
(VA). The study was supported in part by grants from the National Institutes
of Health and VA.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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