Bcl-x(L) Retrotranslocates Bax from the Mitochondria into the Cytosol

Article (PDF Available)inCell 145(1):104-16 · April 2011with92 Reads
DOI: 10.1016/j.cell.2011.02.034 · Source: PubMed
Abstract
The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.
Bcl-x
L
Retrotranslocates Bax
from the Mitochondria into the Cytosol
Frank Edlich,
1
Soojay Banerjee,
1
Motoshi Suzuki,
2
Megan M. Cleland,
1
Damien Arnoult,
4
Chunxin Wang,
1
Albert Neutzner,
3
Nico Tjandra,
2
and Richard J. Youle
1,
*
1
Surgical Neurology Branch, NINDS
2
Laboratory of Molecular Biophysics, NHLBI
National Institutes of Health, Bethesda, MD 20892, USA
3
Universita
¨
tsspital Basel, Augenklinik, Basel 4031, Switzerland
4
INSERM U1014, Hopital Paul Brousse, Villejuif cedex 94807, France
*Correspondence: youle@helix.nih.gov
DOI 10.1016/j.cell.2011.02.034
SUMMARY
The Bcl-2 family member Bax translocates from the
cytosol to mitochondria, where it oligomerizes and
permeabilizes the mitochondrial outer membrane
to promote apoptosis. Bax activity is counteracted
by prosurvival Bcl-2 proteins, but how they inhibit
Bax remains controversial because they neither
colocalize nor form stable complexes with Bax. We
constrained Bax in its native cytosolic conformation
within cells using intramolecular disulfide tethers.
Bax tethers disrupt interaction with Bcl-x
L
in deter-
gents and cell-free MOMP activity but unexpectedly
induce Bax accumulation on mitochondria. Fluores-
cence loss in photobleaching (FLIP) reveals constant
retrotranslocation of WT Bax, but not tethered Bax,
from the mitochondria into the cytoplasm of healthy
cells. Bax retrotran slocation depends on prosurvival
Bcl-2 family proteins, and inhibition of retrotranslo-
cation correlates with Bax accumulation on the
mitochondria. We propose that Bcl-x
L
inhibits and
maintains Bax in the cytosol by constant retrotrans-
location of mitochondrial Bax.
INTRODUCTION
Bcl-2 proteins control many pathways of programmed cell death
in multicellular animals. Members of the Bcl-2 family can be
grouped in prosurvival Bcl-2-like proteins and proapoptotic
Bax-like members (Chipuk and Green, 2008; Cory and Adams,
2002; Youle and Strasser, 2008). The functions of Bcl-2 family
members can be regulated by a diverse group of ‘BH3-only’
proteins that initiate the proapoptotic activities of Bax-like
proteins (Chipuk and Green, 2008).
Bax resides in the cytoplasm of healthy cells and translocates
to the mitochondrial outer membrane (MOM) upon apoptosis
induction (Wolter et al., 1997), where it causes cytochrome c
(cyt c) release from the mitochondrial intermembrane space
and mitochondrial dysfunctions (Gross et al., 1998; Martinou
et al., 1999; Wang et al., 2001; Wei et al., 2001). The three
concomitant events that characterize the commitment of a cell
to apoptosis, Bax oligomerization, cyt c release, and breakdown
of the interconnected mitochondrial network, are tightly linked to
the process of Bax translocation.
An early ‘rheostat model’ proposed that Bax is restrained
by heterodimerization with prosurvival Bcl-2 family proteins
(Korsmeyer et al., 1993). However, this view could not be recon-
ciled with experimental evidence of monomeric Bax residing
in the cytoplasm of healthy cells, in contrast to the mitochondrial
localization of Bcl-2 on the MOM (Hsu et al., 1997; Hsu and
Youle, 1998). Although interactions between Bax and prosurvival
Bcl-2 proteins control Bax activity (Fletcher et al., 2008), the
question remains: How do prosurvival Bcl-2 proteins regulate
Bax from a distance without interacting with Bax in the
cytoplasm?
In an attempt to resolve the dilemma of Bax regulation by pro-
survival Bcl-2 proteins independent of ‘sequestration,’ BH3-
only proteins have been suggested to mediate the link between
cytosolic Bax and the mitochondrial prosurvival proteins. Some
findings indicate that Bax can bind to and be activated by the
BH3-only proteins Bim, Puma, or the proapoptotic Bcl-2 family
protein tBid (Desagher et al., 1999; Kim et al., 2006; Kuwana
et al., 2005; Letai et al., 2002). Accordingly, these Bax ‘activator’’
proteins are proposed to be sequestered and neutralized by pro-
survival Bcl-2 family members in healthy cells. In response to
apoptosis, induction ‘activator’ proteins could be released
from prosurvival Bcl-2 family proteins, perhaps by competition
with other BH3-only proteins binding to prosurvival Bcl-2 family
members, to activate Bax (Kim et al., 2006). Cell-free assays
show a synergistic effect of tBid or Bim on Bax-mediated
membrane permeabilization, suggesting a role of both proteins
in direct Bax activation (Kuwana et al., 2005; Wei et al., 2000).
Apoptosis assays with Bid/Bim DKO MEFs and the phenotypes
of the corresponding knockout mice show that many apoptosis
pathways do not depend on activity of either tBid or Bim (Willis
et al., 2007), whereas the analysis of Bid/Bim/Puma TKO cells
shows an effect on apoptosis induction by several stimuli (Ren
et al., 2010). However, direct binding between Bax and BH3-
only proteins in cells is not readily apparent (Walensky et al.,
2006).
Further evidence indicates that Bax interacts with prosurvival
Bcl-2 proteins and suggests that BH3-only proteins play a role in
104 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
interfering with the heterodimer formation between Bax and pro-
survival Bcl-2 proteins, rather than directly activating Bax (Chen
et al., 2005; Willis et al., 2005, 2007). Bax also has been found to
undergo major conformational changes to integrate in lipid bila-
yers where membrane-bound Bax can form stable complexes
with either tBid or Bcl-x
L
(Dlugosz et al., 2006; Lovell et al.,
2008). However, the models of anti- and proapoptotic Bcl-2
family member interaction fail to explain why during apoptosis
inhibition increased Bcl-x
L
concentrations do not result in an
accumulation of Bax on mitochondria in complex with Bcl-x
L
.
We report here a mechanism of antiapoptotic Bcl-2 family
member inhibition of Bax activation and apoptosis whereby
Bax in the cytoplasm of nonapoptotic cells continually binds to
mitochondria and retrotranslocates back to the cytoplasm
through interaction with Bcl-x
L
.
RESULTS
Disulfide Bonds Constrain the Inactive Bax
Conformation
The activation of Bax involves major changes in its protein
conformation that are linked to mitochondrial localization and
integration into the MOM. We sought to hinder conformational
changes involving a helices 1 and 2 of Bax containing the BH3
motif to analyze their involvement in Bax activity. To constrain
Bax in its inactive conformation, we substituted to cysteine
residues F30 and L63, which are in close proximity, to form an
intramolecular disulfide bond between a helices 1 and 2 (1-2)
(Figure 1A and Figure S1A available online). We also changed
E44 and P130 to cysteines to constrain the flexible loop between
a helices 1 and 2 to the tip of helix 6 (L-6). In addition, the intrinsic
cysteine residues C62 and C126 were substituted by serine
residues (Bax DSH) to avoid interference with the engineered
disulfide bonds.
Previous reports have shown that disulfide bonds can form in
the reducing environment of the cytosol (Bessette et al., 1999;
Locker and Griffiths, 1999; Østergaard et al., 2004; Schouten
et al., 2002). We examined whether the disulfide bonds 1-2
and L-6 are formed in Bax expressed in HCT116 Bax/Bak DKO
cells by SDS-PAGE and western blot in the absence and pres-
ence of b-mercapto-ethanol (BME). Wild-type Bax and the Bax
variants C62S, C126S, and C62/126S (DSH) migrate similarly
with and without BME, whereas Bax variants with one or two
engineered disulfide bonds (1-2, L-6, and 1-2/L-6) migrate faster
in the absence of BME than WT Bax (Figure 1B). The decreased
Stokes radius of the denatured Bax variants in the absence of
BME indicates that the engineered disulfide bonds form in Bax
within cells.
We confirmed the absence of free SH groups in Bax 1-2/L-6 by
thiol trapping using a maleimide derivative with a 10 kDa mPEG
Figure 1. Disulfide Bonds Constrain Bax in Its Inactive Fold
(A) Depiction of the three-dimensional structure of Bax (PDB, 1F16) containing cysteine substitutions for F30, E44, L63, and P130 (red sticks) to form disulfide
bonds constraining a helices 1 (green) and 2 (1-2) and the loop (orange) between a helices 1 and 2 and a helix 6 (L-6) using Pymol software (DeLano Scientific LLC).
Helix 2 is depicted in blue; helix 6, in yellow. See also Figure S1A.
(B) The disulfide bond formation of cysteine GFP-Bax variants in HCT116 Bax/Bak DKO cells was analyzed by SDS-PAGE and western blot of cell extracts in the
presence and absence of b-mercaptoethanol (BME) using rabbit a-GFP antibody. See also Figures S1B–S1D.
(C) Selected spectra of NOESY experiment for Bax 1-2/L-6 and WT Bax. Each strip for the indicated tryptophan side chain labeled above was extracted from the
15
N-edited 3D NOESY spectra. Left panels were obtained from WT Bax, whereas the right panels were from Bax 1-2/L-6. Cross-peaks for NOE interactions for
WT Bax were identified from
15
N-edited 3D NOESY as well as
13
C/
15
N-edited 4D NOESY. Identical strips are displayed for Bax 1-2/L-6, confirming that the fold of
the Bax variant is the same as that of WT Bax. See also Figures S1E and S1F.
Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc. 105
fusion (mPEG-MAL) while WT Bax becomes modified (Fig-
ure S1B). The analysis of Bax variants expressed in HCT116
Bax/Bak DKO cells with mPEG-MAL also showed free SH
groups in GFP-Bax WT that are absent in GFP-Bax DSH (Fig-
ure S1C). Thiol trapping of either GFP-Bax 1-2 or GFP-Bax L-6
shows pools of unmodified but also of modified protein, whereas
GFP-Bax 1-2/L-6 remains unaltered, suggesting stabilization of
a compact Bax fold by the two disulfide bonds, thereby shielding
the disulfides from the reducing environment of the cytosol. The
redox potential of the disulfide bonds of this Bax variant was
determined to be lower than 370 mV (Figure S1D), consistent
with their formation in the cytosol (Schafer and Buettner, 2001).
We analyzed the conformation of recombinant Bax 1-2/L-6 by
NMR in comparison to WT Bax. NMR chemical shift (d) is sensi-
tive to molecular conformation. Differences of chemical shifts
(Dd) between WT Bax and Bax 1-2/L-6 can be used as a probe
of conformational differences of these two molecules. Notice-
able differences in chemical shifts of the backbone amide proton
and nitrogen are present but are limited to the regions where
mutations were introduced (Figure S1 E). The absence of signifi-
cant differences that are not associated with mutations indicates
that the global structure of Bax 1-2/L-6 is essentially the same as
that of WT Bax. In addition, nuclear Overhauser effect (NOE) is
direct evidence of molecular structure, as it reports two protons
within 5 A
˚
. The NOE spectra from five tryptophan side chains
were unaffected by the substitutions (Figure 1C). Noteworthy,
the side chain H31 of Trp158 located at the loop between a6
and a7 helices showed NOEs to Ha and Hg2 of Ile19 that is 11
residues away from the F30C mutation site, where both Ile19
and Cys30 are located within the a1 helix. In WT Bax, the
same NOEs between Trp158 H31 and Ile19 Hg2 and Ha were
observed. We also found that the regions of flexibility of Bax
1-2/L-6 are the same as WT Bax, only differing with reduced
dynamics at the L-6 disulfide tether (Figure S1F). Thus, the intra-
molecular tethers stabilize the native and inactive conformation
in Bax 1-2/L-6 that is similar to inactive WT Bax (see also Supple-
mental Results).
Disulfide Bonds Inhibit Bax Activity and Regulation
by Bcl-x
L
We tested the influence of stabilizing the inactive conformation of
Bax in cells by measuring caspase 3/7 activity. Staurosporine
(STS)-induced caspase activity in HCT116 Bax/Bak DKO cells
expressing Bax DSH is similar to WT Bax-expressing cells
(data not shown) and is prevented by Bcl-x
L
overexpression
(Figure 2A). In parallel to the caspase activity assay in Bax
DSH-expressing cells, STS induces increased cyt c release
and cell death indicated by the release of LDH that is inhibited
by Bcl-x
L
overexpression. Similar activities were obtained in
HCT116 Bax KO cells with Bax DSH or additional single cysteine
substitution of either F30, E44, L63, or P130, showing that the
substitutions used in Bax 1-2/L-6 do not interfere with Bax
activity without disulfide bond formation (data not shown). In all
three assays, Bax 1-2/L-6 lacks STS-inducible activity (Figures
Figure 2. Constraining Bax Prevents Proa-
poptotic Activity and Inhibition by Bcl-x
L
(A) Staurosporine (STS, 1 mM)-induced apoptosis
activity of Bax DSH and Bax 1-2/L-6 based on
caspase 3/7 activity measured in HCT116 Bax/Bak
DKO cells relative to the activity obtained with WT
Bax and normalized to mock-transfected cells.
Results were obtained without (gray) or with (black)
overexpressed Bcl-x
L
. Data represent averages ±
SD; n R 5 3 5 wells.
(B) Cyt c release from the mitochondria analyzed
microscopically in HCT116 Bax/Bak DKO cells
expressing different GFP-Bax variants after
apoptosis induction by 1 mM STS. Results were
obtained without (gray) or with (black) overex-
pressed Bcl-x
L
. Data represent averages ± SD
from triplicates. n R 125 cells.
(C) LDH activity measured after transfecting
HCT116 Bax/Bak DKO cells with different GFP-
Bax variants for 24 hr and inducing apoptosis with
1 mM STS for 16 hr relative to the activity obtained
with cells transfected with GFP-Bax WT and
normalized to mock-transfected cells. Results
were obtained with (black) or without (gray) over-
expressed Bcl-x
L
. Data represent averages ± SD.
n R 4 3 5 wells.
(D) Cyt c release by WT Bax and Bax 1-2/L-6 (see
also Figures S2A and S2B) in absence and pres-
ence of tBid from purified mitochondria. Cyt c is
monitored in the supernatant and pellet by western
blot. VDAC serves as a loading control.
See also Figures S2C and S2D.
106 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
2A–2C). However, in the presence of Bcl-x
L
(Figure 2B) or in
the absence of apoptosis induction (Figures S2A and S2B),
overexpression of Bax 1-2/L-6 induced cyt c release more
than overexpression of WT Bax. The ability of recombinant Bax
1-2/L-6 to induce cyt c release was also tested using mitochon-
dria isolated from Bax/Bak DKO MEFs (Figure 2D). In this assay,
recombinant WT Bax causes the release of cyt c from isolated
mitochondria in the presence of tBid. Recombinant Bax 1-2/
L-6 fails to induce cyt c release even in the presence of tBid.
Thus, the intramolecular tethers in Bax 1-2/L-6 decrease its acti-
vation by BH3-only proteins and regulation by Bcl-x
L
. Although
Bax and Bcl-x
L
do not interact in the cytoplasm of cells, their
interaction can be induced in vitro by detergents (Hsu and Youle,
1997). We assessed whether constraining Bax with intramolecu-
lar tethers interferes with this interaction. Though WT Bax and
Bcl-x
L
interact in the presence of different detergents at concen-
trations greater than CMC, Bax 1-2/L-6 forms heterodimers with
Bcl-x
L
only in Triton X-100, Triton X114, and dodecyl maltoside
(Figure S2C and Supplemental Results). Thus, intramolecular
tethers can interfere with detergent-induced Bcl-x
L
binding.
Tethered Bax Localizes to the Mitochondria
Inactive Bax resides mainly in the cytoplasm. Upon activation,
Bax forms foci at the tips and constriction sites of mitochondria
that is temporally associated with mitochondrial outer
membrane permeabilization (MOMP) and cyt c release (Karbow-
ski et al., 2004). Like WT Bax, Bax DSH is found predominantly in
the cytosol of transfected HCT116 Bax/Bak DKO cells and trans-
locates to mitochondria upon apoptosis stimulation (Figures
3A–3C). Surprisingly, Bax 1-2/L-6 is not located in the cytosol
and smoothly coats the mitochondria in 99% of healthy cells
and remains unchanged in the presence of apoptotic stimuli
(Figures 3A–3C). Whereas Bcl-x
L
overexpression prevents the
localization of Bax DSH to the mitochondria after apoptosis
induction, GFP-Bax 1-2/L-6 circumscribes the mitochondria
even on Bcl-x
L
overexpression (Figure S3A and Supplemental
Results). Cell fractionation confirms that, in contrast to Bax
DSH, most Bax 1-2/L-6 is found in the heavy membrane (HM)
fraction in the absence of apoptosis induction (Figure 3D). Teth-
ered Bax is largely carbonate extractable, suggesting that it
binds mitochondria but does not integrate into the MOM.
Wild-Type, but Not Tethered, Bax Retrotranslocates
from the Mitochondria into the Cytoplasm
Why does tethered Bax localize to mitochondria in healthy cells
despite adopting an inactive conformation? Although WT Bax
resides mainly in the cytoplasm of healthy cells, a fraction
localizes to mitochondria but, in contrast to mitochondrially
embedded Bax found following apoptosis induction, is
carbonate extractable (Desagher et al., 1999). We hypothesized
that the mitochondrial Bax pool could be in equilibrium with
cytosolic Bax in healthy cells, which could be disrupted by Bax
tethers. In an attempt to distinguish between cytosolic and mito-
chondrial Bax and compare WT Bax with Bax 1-2/L-6, we per-
formed fluorescence loss in photobleaching (FLIP) with different
GFP-Bax variants expressed in HCT116 Bax/Bak DKO cells. To
this end, we repeatedly bleached a region in the nucleus of
a transfected cell (Figure 4A, white square). The declining GFP
fluorescence in the targeted cell was followed by assigning
regions of interest in the cytoplasm (green circle) and on the
mitochondria (red and blue circles) in Figure 4A. GFP-Bax readily
crosses the nuclear envelope, and cytosolic GFP fluorescence of
the targeted cell was bleached rapidly by FLIP, whereas the
neighboring reference cell fluorescence (black circle) remained
stable, ruling out photobleaching during imaging (Figures 4A
and 4B). After reducing the cytosolic GFP-Bax signal, the mito-
chondrial GFP-Bax pool was readily apparent (Figure 4B,
arrows). The decay of mitochondrial GFP-Bax fluorescence by
FLIP occurs within 660 s following a first-order kinetic at a rate
(4.7 ± 0.1 3 10
3
s
1
) that is notably slower than the loss in cyto-
solic fluorescence (Figures 4B and 4C and Figure S4A). Interest-
ingly, Bcl-x
L
overexpression causes more than an 80% increase
in the rate of mitochondrial fluorescence reduction during FLIP at
comparable levels of Bax expression (Figures 4B–4E). The loss in
mitochondrial GFP-Bax fluorescence during FLIP suggests that
Bax could exist in an equilibrium between mitochondrial and
cytosolic states (Figure 4D).
The presence of MG132 had no effect on GFP-Bax fluores-
cence loss with or without Bcl-x
L
(Figure S4B), indicating that
proteasomal degradation does not account for the decrease in
mitochondrial fluorescence during FLIP. To directly assess Bax
return to the cytosol from mitochondria, we analyzed fluores-
cence recovery after photobleaching (FRAP) of cytosolic GFP-
Bax (Figure S4C). Following the bleach, GFP-Bax fluorescence
increases in the cytosol by about 25% after 400 s following
a first-order kinetic (Figure 4F). Overexpression of Bcl-x
L
increases the cytosolic reappearance of GFP-Bax fluorescence
more than 2-fold when mitochondrial postbleach GFP-Bax levels
were comparable (Figure S4D). We examined whether continual
retrotranslocation is balanced by continual binding of Bax to
mitochondria in healthy cells. By photobleaching half of a cell
expressing GFP-Bax (Figure 4H), we quantified the binding of
Bax to mitochondria over the subsequent 10 min (Figures 4G
and 4H and Figure S4E). Bax WT translocates to mitochondria
at a rate of 4.7 ± 0.2 3 10
3
s
1
, consistent with an equilibrium
between on and off rate.
Although FLIP analyses appear to measure an increase in
mitochondrial Bax off rates by Bcl-x
L
, it could be suggested
that WT Bax and Bcl-x
L
may compete for the same binding
site on the mitochondria, causing increased Bax retrotransloca-
tion into the cytoplasm. This possibility was tested by analyzing
the effect of untagged Bax overexpression on GFP-Bax retro-
translocation (Figure S4F). In contrast to Bcl-x
L
overexpression,
Bax slightly decreases the GFP-Bax retrotranslocation rate
(4.4 ± 0.1 3 10
3
s
1
), indicating no competition between Bax
and Bcl-x
L
for MOM binding. In the presence of untagged Bax,
the overexpression of Bcl-x
L
accelerates GFP-Bax retrotranslo-
cation (7.0 ± 0.2 3 10
3
s
1
) but significantly less than without
untagged Bax (8.6 ± 0.4 3 10
3
s
1
), suggesting that Bax can
compete with GFP-Bax for Bcl-x
L
-mediated retrotranslocation.
Mitochondrial Bax retrotranslocation into the cytoplasm depen-
dent on the Bcl-x
L
concentration may provide a rationale for the
mitochondrial accumulation of Bax 1-2/L-6. The intramolecular
tethers in Bax 1-2/L-6 may interfere with Bcl-x
L
-mediated retro-
translocation, as they also disrupt the interaction between Bax
and Bcl-x
L
in some detergents. We applied FLIP to analyze
Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc. 107
Figure 3. Bax 1-2/L-6 Localizes to the Mitochondria
(A) Confocal imaging of HCT116 Bax/Bak DKO transfected with GFP-Bax DSH or GFP-Bax 1-2/L-6 with or without treatment with 1 mM actinomycin D (ActD) for
2 hr. Q-VD was used to prevent caspase activation. GFP-fluorescence is depicted in the second panels and in green in the merge and detail, whereas a-Tom20
staining is shown in the left panels and in red in the merged and detail images. In the merged and detail images, colocalization is shown in yellow. The white line in
the lower-right corner of every image is the scale of 10 mm. White broken lines in the merge images show the section analyzed in the line scans (B). The merged
section depicted in the detail panel is indicated by a white box.
(B) Line scans show the fluorescence intensities of GFP-Bax signals (green) and mitochondria stained by a-Tom20 staining (red) along the selected line (A) in cells
transfected with GFP-Bax DSH or Bax 1-2/5-6 either with or without ActD treatment.
(C) Quantification of confocal images of HCT116 Bax/Bak DKO cells transfected with either GFP-Bax DSH or 1-2/L-6 showing predominantly cytosolic Bax
cell populations in percent of the total cell population after 2 hr treatment with 1 mM ActD with or without Bcl-x
L
coexpression. Data represent averages of
triplicates ± SD; n R 150 cells. See also Figure S3.
108 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
Bax 1-2/L-6 retrotranslocation, bleaching the low GFP-Bax 1-2/
L-6 fluorescence in the cytoplasm, as was done for WT GFP-
Bax. Mitochondrial GFP-Bax 1-2/L-6 fluorescence intensity
was not significantly reduced by repeated bleaching (Figures
5A and 5B). In contrast to WT Bax, Bcl-x
L
overexpression did
not detectably increase the retrotranslocation of Bax 1-2/L-6 in
a 660 s time frame (Figures 5A and 5B). Thus, Bax 1-2/L-6 is defi-
cient in retrotranslocation. We examined the role of helix 9 in Bax
1-2/L-6 binding to mitochondria. Bax 1-2/L-6 displayed the
same sensitivity to S184 mutations as WT Bax (Nechushtan
et al., 1999)(Figures S5 A and S5B), indicating that helix 9 is
required for Bax 1-2/L-6 binding to mitochondria.
Bax Retrotranslocation Depends on BH3 Interactions
with Prosurvival Bcl-2 Proteins
We tested the effect of different Bcl-2 family members on Bax
retrotranslocation. Overexpression of Bcl-2 and Mcl-1 acceler-
ated Bax retrotranslocation similarly to Bcl-x
L
(Figure 6A and Fig-
ure S6A). In contrast, the BH3-only protein Bim reduced the rate
of Bax retrotranslocation more than 3-fold to 1.3 ± 0.2 3 10
3
s
1
in HCT116 Bax/Bak DKO cells that did not contain Bax foci.
Endogenous Bak expression tested by comparing HCT116
Bax/Bak DKO and Bax KO cells has no influence on Bax retro-
translocation (Figure 6A and Figure S6A). After MOMP or in the
presence of the viral Bax inhibitor vMIA (Arnoult et al., 2004),
WT Bax retrotranslocation is inhibited (see also Supplemental
Results and Figures S6B–S6E).
To analyze whether binding of prosurvival Bcl-2 proteins to
Bax is required to mediate Bax retrotranslocation, we examined
Bcl-x
L
G138A, a variant that is deficient in Bax binding
and apoptosis inhibition (Desagher et al., 1999; Dlugosz
et al., 2006; Sedlak et al., 1995). In contrast to WT Bcl-x
L
,
G138A failed to accelerate retrotranslocation of GFP-Bax when
expressed at levels comparable to WT Bcl-x
L
(Figures 6 B and
6C). Furthermore, the Bcl-2/Bcl-x
L
inhibitor ABT-737 (Oltersdorf
et al., 2005) reduced the rate of Bax retrotranslocation by
more than 75%, suggesting that endogenous Bcl-2 family
members mediate Bax retrotranslocation (Figure 6D and
Figure S7A). These results indicate the involvement of direct
interactions between prosurvival Bcl-2 proteins and Bax for
retrotranslocation.
The Bax variant D68R has been previously shown to exhibit
insensitivity toward Bcl-2/Bcl-x
L
inhibition and potent proapop-
totic activity (Fletcher et al., 2008). Interestingly, Bax D68R
constitutively localizes to the mitochondria of HCT116 Bax/
Bak DKO cells in the absence of apoptosis stimuli (Figures 6E
and 6F). Bax D68R localizes to the mitochondria even in cells
not displaying cyt c release (Figure 6F). We analyzed whether
Bax D68R retrotranslocation could be accelerated by overex-
pression of the prosurvival Bcl-2 proteins Bcl-2, Bcl-x
L
, and
Mcl-1. Bax D68R retrotranslocates at less than half the rate of
WT Bax (Figure 6G), whereas the S184V substitution in helix 9,
which also increases the mitochondrial Bax pool, only slightly
decreases Bax retrotranslocation (see also Supplemental
Results and Figures S7B and S7C). In contrast to WT Bax, the
retrotranslocation rate of D68R is only slightly increased by
Bcl-2 and Bcl-x
L
overexpression from 2.1 ± 0.1 3 10
3
s
1
to
about 3.9 3 10
3
s
1
, whereas overexpression of Mcl-1 does
not accelerate Bax D68R retrotranslocation (Figure 6G). The
ability of the different prosurvival Bcl-2 proteins to increase
Bax D68R retrotranslocation correlates with the relative affinities
of Mcl-1, Bcl-2, and Bcl-x
L
for Bax D68R (Fletcher et al., 2008).
The diminished retrotranslocation of Bax D68R extends the
results obtained with tethered Bax 1-2/L-6, indicating the impor-
tance of prosurvival Bcl-2 protein interactions with the BH3
domain of Bax, which is further indicated by the retrotransloca-
tion of a Bcl-x
L
chimera with its helices 2 and 3 replaced by the
corresponding Bax helices (George et al., 2007). The retrotrans-
location rate of this chimera is similar to the rate of Bax
(Figure S7D).
The possibility of Bcl-x
L
retrotranslocation was analyzed by
performing FLIP with HCT116 Bax/Bak DKO cells expressing
GFP-Bcl-x
L
. GFP-Bcl-x
L
localizes in these cells predominantly
to the mitochondria (Figure S7E) and retrotranslocates in the
absence of Bax with a low rate (1.4 ± 0.1 3 10
3
s
1
) from the
mitochondria into the cytoplasm ( Figure 6H). Overexpression
of Bax accelerates Bcl-x
L
retrotranslocation about 3.5-fold, sug-
gesting that they interact on mitochondria, retrotranslocate
together, and dissociate in the cytosol. Interestingly, ABT-737
increases the Bcl-x
L
retrotranslocation rate (Figure S7F).
Bax 1-2/L-6 Adopts a 6A7-Positive Fold
on the Mitochondria
Upon translocation to the mitochondria during apoptosis, WT
Bax exposes an epitope consisting of P13-I19 at the N terminus
of helix 1 for the monoclonal antibody 6A7 that is not accessible
in cytosolic and mitochondrial WT Bax in healthy cells (Hsu and
Youle, 1998). This change in the 6A7 epitope correlates with foci
formation and cyt c release (Nechushtan et al., 1999). Despite
constitutive mitochondrial localization, Bax 1-2/L-6 fails to form
foci.
Surprisingly, Bax 1-2/L-6 is 6A7 positive in some, but not all,
cells (Figures 7A and 7B) while circumscribing the mitochondria
(Figure 3A). Only a subset of Bax 1-2/L-6 on the mitochondria
adopts a 6A7-positive fold as inferred by the Pearson’s coeffi-
cient of about 0.7 (Figure 7C). The pool of 6A7-positive cells
transfected with Bax 1-2/L-6 is slightly decreased by Bcl-x
L
overexpression, whereas almost 100% of WT Bax-expressing
cells are 6A7 negative with Bcl-x
L
overexpression (Figure 7B).
Interestingly, Bax 1-2/L-6 changes to its 6A7-positive conforma-
tion gradually over 24 hr on the mitochondria of healthy cells
(Figure 7D).
Although the disulfide tethers in Bax 1-2/L-6 would reduce the
conformational flexibility of its N-terminal part, they do not
completely block Bax from undergoing a conformational change
on the mitochondria that results in the exposure of the 6A7
(D) GFP-Bax WT and GFP-Bax 1-2/L-6 localization in HCT116 Bax/Bak DKO cells analyzed by SDS-PAGE and western blot after fractionation into cytosol (C) and
heavy membrane fraction (HM) and subjecting membrane-bound proteins to carbonate extraction and analyzing pellet (P) and supernatant (S) with rabbit a -GFP,
rabbit a-GAPDH, mouse a-cyt c, and rabbit a-Tom20 antibodies.
Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc. 109
Figure 4. Wild-Type Bax Retrotranslocates from the Mitochondria into the Cytoplasm
(A) GFP-Bax fluorescence is monitored in a FLIP experiment with 15 bleachings at 488 nm in the region marked as square. Changes in Bax fluorescence on the
mitochondria are detected in two areas (red circle and blue circle, respectively), and an additional area monitors changes in the cytosolic fluorescence (green
circle), whereas a ROI measurement in the neighboring cell serves as a control for cell-specific bleaching (black circle).
(B) FLIP of GFP-Bax in the absence (top) and presence (bottom) of overexpressed Bcl-x
L
diminishes GFP-Bax fluorescence in the cytoplasm of both targeted
cells (circled) completely after 90 s, and GFP fluorescence is detected only on the mitochondria (arrows). The mitochondrial GFP-Bax signal in the presence of
overexpressed Bcl-x
L
is lower at 90 s. Time points in seconds are displayed above the pictu res.
(C) Bcl-x
L
increases the rate of Bax retrotranslocation. FLIP of mitochondrial GFP-Bax in the absence (circle) and presence (filled triangle) of overexpressed Bcl-
x
L
. Fluorescence of the neighboring cell is shown as control (open triangle). Data represent averages ± SEM from 20 ROI measurements per condition. See also
Figure S4A.
(D) Prior to FLIP, GFP-Bax localizes to mitochondria and cytosol (i). FLIP bleaches cytosolic Bax (ii), but in addition, the mitochondrial fluorescence is diminished
because bleac hed Bax molecules translocate to the mi tochondria while fluorescent GFP-Bax retrotranslocates into the cytoplasm dependent on Bcl-x
L
(iii). After
an extended time of 15 FLIP iterations, all GFP-Bax molecules are bleached (iv).
110 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
epitope. Because Bax 1-2/L-6 does not show induced apoptotic
activity, the 6A7-positive conformational change smoothly
coating mitochondria seems to be an intermediate step en route
to activation, likely correlating with spontaneous induction of cyt
c release (Figures S2A and S2B) upstream of foci formation. As
WT Bax does not reach the 6A7-positive state when circum-
scribing mitochondria in healthy cells, the prolonged association
of Bax 1-2/L-6 with mitochondria may be the stimulus. Subse-
quent conformational rearrangements inhibited by the tethers
likely are associated with foci formation.
DISCUSSION
Prosurvival Bcl-2 proteins prevent apoptosis by inhibiting Bax
and Bak. They block Bax translocation from the cytosol to the
mitochondria, Bax oligomerization, and MOMP. Paradoxically,
prosurvival Bcl-2 proteins on the mitochondria stabilize Bax
localization in the cytosol, without forming stable heterodimeric
complexes. Bax regulation by Bcl-2 thus creates a spatial
paradox that has been addressed by previous models of Bax
activation (Kim et al., 2006; Willis et al., 2007).
(E) Similar levels of GFP-Bax WT and GFP-Bax 1-2/L-6 expression in HCT116 Bax/Bak DKO cells in the presence and absence of Bcl-x
L
overexpression analyzed
by SDS-PAGE and western blot using rabbit a-GFP, mouse a-Bcl-x
L
, and rabbit a-Tom20 antibodies.
(F) GFP-Bax fluorescence is recovering in the cytoplasm after a single bleac h at 488 nm (inset shows magnification), and Bcl-x
L
is increasing the rate of this
fluorescence intensity regain, consistent with the FLIP experiments. Data represent averages ± SEM from 22 (Bcl-x
L
) and 16 (+Bcl-x
L
) ROI measuremen ts. See
also Figures S4C and S4D.
(G) Translocation of Bax to the mitochondria of healthy cells analyzed by cell bleaching (Figure S4E). Recovery of mitochondrial GFP-Bax WT fluorescence 1, 2, 4,
and 10 min after bleach was compared in the absence (red, open circles) or presence (blue, filled circles) of Bcl-x
L
to unbleached mitochondria in 12 different cells
per data point ± SD.
(H) HCT116 Bax/Bak DKO cells expressing GFP-Bax WT imaged before the analysis by bleaching (first and third panels from the left; see also Figure S4E). Then
the cells (circled) were bleached in the area in the red squares. After 1 or 10 min, the fluorescence in the cytoplasm was bleached and the cells were imaged
(second and forth panels, respectively) for analysis (in G).
See also Figures S4B and S4F.
Figure 5. Bax 1-2/L-6 Is Deficient in Retrotranslocation
(A) Time course recorded for GFP-Bax 1-2/L-6 in the absence (top) and presence (bottom) of overexpressed Bcl-x
L
in FLIP experiments. FLIP diminishes GFP-
Bax 1-2/L-6 in the cytoplasm of a targeted cell (circled) quickly in parallel to WT Bax, but mitochondrial signals (arrows) remain stable. Time points in seconds
during FLIP iterations are displayed above the pictures. See also Figure S5.
(B) FLIP of mitochondrial GFP-Bax 1-2/L-6 as shown in (A) in the absence (blue, filled circles) and presence (red, filled triangles) of overexpressed Bcl-x
L
. The
neighboring cell fluorescence (open circles) and cells transfected with GFP-Bax WT (filled circles) serve as controls. Data repres ent averages ± SEM from 20
(Bax 1-2/L-6 Bcl-x
L
) and 18 (Bax 1-2/L-6 + Bcl-x
L
) ROI measurements.
Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc. 111
Figure 6. Bax Retrotranslocation Depends on Interactions between BH3 and Prosurvival Bcl-2 Proteins
(A) Influence of Bax interaction partners on retrotranslocation. Prosurviva l and proapoptotic Bax interaction partners are displayed as a schematic depiction of
their Bcl-2 homology domain (BH) organization, with the C-terminal transmembrane domains (TM) on the right. Bax retrotranslocation rates in the presence and
absence of overexpressed Bcl-2, Bcl-x
L
, Mcl-1, or Bim measured in HCT116 Bax/Bak DKO or in HCT116 Bax KO for measurements in the presence of
endogenous Bak are depicted on the right in %. See also Figure S6A.
(B) Interactions between Bcl-x
L
and Bax are implicated in retrotranslocation. FLIP of GFP-Bax in the absence (filled circles) or presence of Bcl-x
L
WT (dashed line)
or Bcl-x
L
G138A (red, open circles). The neighboring cell fluorescence serves as control (line). Data display averages ± SEM from 12 ROI measurements per
condition.
(C) Similar expression levels of Bcl-x
L
variants in HCT116 Bax/Bak DKO cells analyzed by SDS-PAGE and western blot using mouse a-Bcl-x
L
and rabbit a-GFP to
probe for GFP-Bax overexpression and rabbit a-Tom20 antibodies.
(D) FLIP experiment with GFP-Bax in absence (filled circles) and presence (dashed line) of overexpressed Bcl-x
L
or Bcl-x
L
inhibitor ABT-737 (red, open circles).
The fluorescence of the neighboring cell serves as control (line). Data represent averages ± SEM from 24 (+ABT-737) and 10 (+DMSO) ROI measurements. See
also Figure S7A.
112 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
We propose a model of continuous Bax retrotranslocation
from mitochondria that is consistent with results from numerous
labs. We find that Bax translocates constantly to the mitochon-
dria in healthy cells, where prosurvival Bcl-2 proteins, such as
Bcl-x
L
, bind Bax and retrotranslocate it back into the cytoplasm,
thereby stabilizing the inactive Bax conformation (Figures 7E
and 7F). Bcl-x
L
and Bax both retrotranslocate from mitochondria
and accelerate the rate of each other’s retrotranslocation
after transient interaction on mitochondria, perhaps through
trans-sequestration of the C-terminal tails (Jeong et al., 2004).
Evidence for direct interaction is based on the inhibition of Bax
retrotranslocation when the Bax-Bcl-x
L
binding is disrupted by:
(1) the G138A mutation in the hydrophobic groove of Bcl-x
L
(Sedlak et al., 1995), (2) the D68R mutation in the BH3 domain
of Bax (Fletcher et al., 2008), and (3) the Bcl-x
L
inhibitor ABT-
737 (Oltersdorf et al., 2005). The interaction between Bax and
Bcl-x
L
requires prior conformational changes in the N-terminal
part of Bax because preventing these conformational changes
by intramolecular tethers disrupts interaction with Bcl-x
L
in
detergents and Bax retrotranslocation.
The absence of retrotranslocation results in Bax 1-2/L-6 accu-
mulation on the mitochondria in healthy cells. Wild-type Bax,
however, only accumulates on mitochondria when the activities
of prosurvival Bcl-2 proteins are blocked by BH3-only proteins,
such as Bim, or by ABT-737. Bax accumulated on mitochondria
upstream of MOMP can dissipate by retrotranslocation if pro-
survival Bcl-2 proteins become available again, as observed
when cells reattach to substrate following transient anoikis
(Gilmore et al., 2000).
Conformational changes of Bax on the mitochondria during
apoptosis involve the N terminus of Bax and can be detected
using the monoclonal antibody 6A7. Despite its reduced
apoptotic activity, tethered Bax eventually adopts a 6A7-positive
fold but does not form mitochondrial foci. Although in cell-free
assays tethered Bax completely lacks tBID activated MOMP,
consistent with the lack of apoptosis-induced activation in cells,
tethered Bax can spontaneously induce some degree of MOMP
within cells even in the presence of Bcl-x
L
, likely through this
6A7-positive form. Because the 6A7 antibody can compete for
Bcl-x
L
binding to Bax (Hsu and Youle, 1998), a 6A7-positive
conformation of WT Bax may normally exist, circumscribing
mitochondria that remains undetectable because 6A7 binding
is sterically blocked by Bcl-x
L
bound to Bax. Bax conformational
changes in a helices 1 and 2 could be a normal consequence of
Bax binding to the mitochondria perhaps stimulated by lipid
interactions (Kuwana et al., 2002). If not retrotranslocated, mito-
chondrial WT Bax becomes active due to further conformational
changes and oligomerization to cause MOMP (Figure 7F).
In addition to a reduced Bax retrotranslocation (off rate), mito-
chondrial Bax accumulation could also result from an increase in
the Bax translocation (on rate), which may depend on direct Bax
activation by BH3-only proteins (Kuwana et al., 2005). Even the
steady-state binding of Bax to mitochondria in healthy cells
may result from the activity of residual levels of BH3-only
proteins in healthy cells. Bax binding to the MOM appears to
be influenced by the exposure of the C-terminal membrane
anchor (Gavathiotis et al., 2010), which may also depend on
isomerization of the prolyl bond preceding P168 and its acceler-
ation by the PPIase Pin1 (Shen et al., 2009). Bax translocation to
the MOM, however, seems not to be influenced by Bcl-x
L
.
Despite the robust interaction of Bax and Bcl-x
L
in detergents
(Hsu and Youle, 1998) and in membranes (Dlugosz et al., 2006),
increased concentrations of prosurvival mitochondrial-bound
Bcl-2 proteins in cells do not result in Bax accumulation on mito-
chondria. In contrast, Bax can be directly bound and inhibited
by the viral protein vMIA that accumulates Bax on the mitochon-
dria as it inhibits apoptosis (Arnoult et al., 2004). In healthy cells,
the subcellular location of Bax depends on constant retrotrans-
location of mitochondrial Bax into the cytosol by prosurvival
Bcl-2 proteins. Minimization of a mitochondrial Bax pool that is
susceptible for activation is likely to prevent apoptosis and
explains the spatial paradox of Bcl-2 protein inhibition of Bax.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfection
HCT116 cells were cultured in McCoy’s 5A medium supplemented with 10%
heat-inactivated fetal bovine serum and 10 mM HEPES in 5% CO
2
at 37
C.
HCT116 Bax/Bak DKO cells were obtained by deletion of the Bak gene by
homologous recombination in the HCT116 Bax
/
cells (C.W. and R.J.Y. ,
unpublished data). Cells were transfected with PolyJet (SignaGen) or Lipofect-
amine LTX (Invitrogen) typically with 100 ng of the GFP-Bax construct accord-
ing to the manufacturer’s instructions, and cells were incubated for 6–8 hr
for confocal imaging. For western blot, cells were harvested 8 hr after
transfection.
Confocal Microscopy
HCT116 Bax/Bak DKO cells were seeded on a chambered coverglass (Thermo
Scientific) in McCoy’s 5A medium (10 mM HEPES), grown for 20 hr, trans-
fected, and incubated for 6–8 hr. The cells were then fixed with 4% paraformal-
dehyde solution for 10 min and washed with PBS. The fixed cells were
permeabilized with Triton X-100 for 15 min at room temperature. For double -
immunofluorescence staining, cells were first incubated with 5% BSA in
PBS for 1 hr at room temperature, followed by incubation with appropriate
primary antibodies (anti-Tom20 and anti-6A7 antibodies) in 5% BSA solution
for 2 hr, and probed with an Alexa-594- and Alexa-647-conjugated secondary
antibody (Invitrogen). Confocal analysis was performed on a Zeiss 510 META
confocal LSM microscope equipped with argon (458/488/514 nm lines) and
HeNe (543/633 nm) lasers. For live cell experiments measuring the recovery
(E) GFP-Bax WT and GFP-Bax D68R localization in HCT116 Bax/Bak DKO cells analyzed by SDS-PAGE and western blot after fractionation into cytosol (C) and
heavy membrane fraction (HM) with rabbit a-GFP, rabbit a-GAPDH, and rabbit a-Tom20 antibodies.
(F) Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bax D68R (center, green in the merge) and stained for cyt c (left, red in the merge).
Colocalization between Bax D68R and cyt c is shown as yellow in the merge (right) when cyt c is not released from the mitochondria without apoptotic stimuli. The
white line in the lower-right corner of every image is the scale of 10 mm.
(G) Retrotranslocation rates measured for Bax WT and Bax D68R in the absence (black) and presence of Mcl-1 (gray), Bcl-2 (dark gray), or Bcl-x
L
(light gray). Data
represent averages ± SD. See also Figures S7B and S7C.
(H) FLIP analysis of GFP-Bcl-x
L
in the absence (red, open circles) and presence (blue, filled triangles) of overexpressed Bax (see also Figures S7E and S7F). The
fluorescence of the neighboring cell serves as control (line). Data represent averages ± SEM from 15 (Bax) and 15 (+Bax) ROI measurements.
See also Figures S6B–S6E and Figure S7D.
Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc. 113
Figure 7. Mitochondrial Bax 1-2/L-6 Is 6A7 Positive
(A) Confocal imaging of HCT116 Bax/Bak DKO cells transfected with GFP-Bax 1-2/L-6 in the presence and absence of ActD (1 mm) treatment for 2 hr. Q-VD was
used to prevent caspase activation. a-6A7 staining is shown in the left panels or in red in the merged and detailed pictures on the right. GFP-Bax 1-2/L-6 is
depicted in the second panels or in green in the merge and detail panels, where colocalization is shown in yellow. The white lines show the scale of 10 mm. The
merged section depicted in the detail panel is indicated by a white box.
(B) a-6A7 staining is quantified in HCT116 Bax/Bak DKO cells expressing GFP-Bax DSH or GFP-Bax 1-2/L-6 with or without Bcl-x
L
overexpression. Data
represent averages from triplicates ± SD; n R 150 cells. p values according the unpaired Student’s t test for the comparison with Bax WT in the absence of
overexpressed Bcl-x
L
are depicted.
(C) Comparison of the Pearson’s coefficient for the colocalization between a-6A7 staining and GFP fluorescence in HCT116 Bax/Bak DKO cells transfected with
either GFP-Bax DSH or 1-2/L-6. The confidence range is depicted as box ± SD with the mean (line) of the data set. Dots represent the most extreme data points
for GFP-Bax 1-2/L-6. n R 10 cells. The p value for both data sets according to the unpaired Student’s t test is depicted.
(D) Time-dependent changes in a-6A7 staining monitored by confocal imaging in HCT116 Bax/Bak DKO cells expressing GFP-Bax 1-2/L-6 in % of total
expressing cell population. Individual measurements are displayed as open circles with the mean shown as a black circle ± SD.
(E) Bax (red) and Bcl-x
L
(blue) constantly translocate to the mitochondria and coretrotranslocate back into the cytosol, stabilizing cytosolic Bax in healthy cells.
Retrotranslocation requires a conformational change in Bax.
(F) In the absence of free Bcl-x
L
, mitochondrial Bax may undergo further conformational changes that can lead to Bax activity or integration into the membrane.
114 Cell 145, 104–116, April 1, 2011 ª2011 Elsevier Inc.
after FRAP, one ROI within the nucleus of a cell of interest was photobleached
with the argon laser at 100% intensity. Recovery of fluorescence in the cyto-
plasm was monitored immediately after photobleaching by imaging the
bleached cell in 20 s intervals with low laser intensity (1%). The results were
normalized setting the starting fluorescence to 100% signal.
For Bax translocation assay, the cells were incubated with mitotracker-far
red for 10 min prior to analysis. Approximately half of an analyzed cell was
bleached with high laser powe r (100%) for 17.5 ms. After either 1, 2, 4, or 10
min, the cytoplasm of the cell was bleached a second time for 25 ms with
high laser power. After the bleaching, two different ROI each were assigned
for unbleached and bleached mi tochondria.
FLIP
In FLIP experiments, a single spot with a diameter of 1 mm within the nucleus
was repeatedly bleached with two iterations of 100% power of a 488 nm laser
line (100% output) using a Zeiss LSM510 META with 633 PlanFluor lens. The
average diameter of a single z axis plane varied between 2 and 2.5 mm. Two
images were collected after each bleach pulse, with 30 s between bleach
pulses. After collecting 30 images, two separate measurements on the mito-
chondria were taken to analyze the fluorescence loss. Unbleached control
cells were monitored for photobleaching due to image acquisition. The rate
of loss in fluorescence on the mitochondria was calculated from fluorescence
intensity measurements using the Zeiss LSM software. The fluorescence
intensities were normalized by setting the initial fluorescence to 100% signal.
Plots are shown as normalized fluorescence over time.
Apoptosis Activity Assays
For caspase 3/7 measurements, HCT116 Bax/Bak DKO cells were transfected
with different Bax constructs in 96-well plates and incubated with or without
1 mM STS for 4 hr. Then, Apo-ONE caspase 3/7 Reagent (Promega) was added
according to manufacturer’s protocol. The samples were incubated for 16 hr in
the dark and then analyzed by measuring the fluorescence with an excitation
wavelength of 488 nm and an emission wavelength range of 530 nm. For LDH
measurements, 96-well plates with HCT116 Bax/Bak DKO cells transfected
with different Bax constructs were incubated with 1 mM STS for 24 hr. Then,
50 ml of the supernatant from each well was transferred in a new plate, and
50 ml of the substrate mix (Promega, cytotox 96 kit) was added to each well
of the plate. After 30–60 min, 50 ml of stop solution was added, and the absor-
bance at 490 nm was detected.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Results, Extended Experimental
Procedures, seven figures, and four movies and can be found with this article
online at doi:10.1016/j.cell.2011.02.034.
ACKNOWLEDGMENTS
We thank Dr. D.W. Andrews and Dr. J.-C. Martinou for helpful discussions and
comments. We also thank Dr. D.R. Green for the Omi-mCherry construct. This
work is supported by the Leopoldina, National Academy of Sciences,
Germany (F.E.); La Ligue contre le Cancer (D.A.); the NHLBI (M.S. and N.T.);
and the NINDS intramural programs.
Received: September 8, 2010
Revised: December 28, 2010
Accepted: February 15, 2011
Published: March 31, 2011
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    • "Following incubation, cells were immunostained for Bax or Bcl-xL, and analysis of Bax/Bcl-xL translocation to the mitochondria was done. To assess colocalization between immune-labeled Bax/Bcl-xL and green fluorescent protein–expressing mitochondria, the method of Edlich et al. (2011) Animal model, passive Heymann nephritis Animals were divided into a control group and a PHN group. PHN was induced by a single injection of rabbit anti-Fx1A antibody (a kind gift from Professor David J. Salant, Boston University Medical Center), control rats were given vehicle. "
    [Show abstract] [Hide abstract] ABSTRACT: There is a great need for treatment that arrests progression of chronic kidney disease. Increased albumin in urine leads to apoptosis and fibrosis of podocytes and tubular cells and is a major cause of functional deterioration. There have been many attempts to target fibrosis, but because of the lack of appropriate agents, few have targeted apoptosis. Our group has described an ouabain-activated Na,K-ATPase/IP3R signalosome, which protects from apoptosis. Here we show that albumin uptake in primary rat renal epithelial cells is accompanied by a time- and dose-dependent mitochondrial accumulation of the apoptotic factor Bax, down-regulation of the antiapoptotic factor Bcl-xL and mitochondrial membrane depolarization. Ouabain opposes these effects and protects from apoptosis in albumin-exposed proximal tubule cells and podocytes. The efficacy of ouabain as an antiapoptotic and kidney-protective therapeutic tool was then tested in rats with passive Heymann nephritis, a model of proteinuric chronic kidney disease. Chronic ouabain treatment preserved renal function, protected from renal cortical apoptosis, up-regulated Bax, down-regulated Bcl-xL, and rescued from glomerular tubular disconnection and podocyte loss. Thus we have identified a novel clinically feasible therapeutic tool, which has the potential to protect from apoptosis and rescue from loss of functional tissue in chronic proteinuric kidney disease.
    Article · May 2016
    • "This observation, combined with the membrane-dependent homo-oligomerization and the constitutive activities of Bax/Bak in these cells, eliminates the theoretical need for a " direct activator " outside the closed system of Bax/Bak–OMM. The constitutive OMM localization of Bax in Bcl-2 allKO cells is consistent with results from Youle and coworkers (Edlich et al. 2011) that demonstrated that Bax has a tendency to constantly move to the OMM in healthy cells and that the prosurvival Bcl-2 proteins, such as BclxL , possess a " retrotranslocation activity, " shuttling Bax from the OMM to the cytosol. Interestingly, Bak was also found to be retrotranslocated by Bcl-xL, albeit at a much slower rate (Todt et al. 2015 ). "
    [Show abstract] [Hide abstract] ABSTRACT: The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the anti-apoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM), and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.
    Full-text · Article · Apr 2016
    • "There are at least two distinct mechanisms whereby BCL2-like proteins mediate such an effect: (1) by physically interacting with BAX and BAK1 and hence preventing their oligomerization [37] ; and (2) by sequestering other members of the Bcl-2 protein family that activate BAX and BAK1 in response to stress, the so-called " BH3-only proteins " [38]. Moreover, BCL-X L has been attributed the capacity to retrotranslocate active BAX to the cytosol (where it normally resides in its inactive state) [39]. Importantly, MOMP drives intrinsic apoptosis not only as it initiates the apoptosome-dependent activation of CASP3 (which cleaves several substrates that are important for cellular survival), but also because it entails the immediate dissipation of the mitochondrial transmembrane potential (Δψ m , which is required for ATP synthesis and several other mitochondrial functions) [40, 41]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondria are fundamental for eukaryotic cells as they participate in critical catabolic and anabolic pathways. Moreover, mitochondria play a key role in the signal transduction cascades that precipitate many (but not all) regulated variants of cellular demise. In this short review, we discuss the differential implication of mitochondria in the major forms of regulated cell death.
    Full-text · Article · Mar 2016
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