Department of Biochemistry and Molecular Biology, PO Box 13D, Monash University, Victoria 3800, Australia
* Author for correspondence (e-mail: phillip.nagley{at}med.monash.edu.au)
Accepted 12 December 2002
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Summary |
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Key words: Apoptosis, Mitochondria, Photosensitisation, Confocal microscopy, Chloromethyl-X-Rosamine
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Introduction |
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An intriguing general question is whether communication between the
multiple mitochondria within individual cells occurs to cooperatively enhance
the signalling role of these organelles in apoptosis. Concepts of
communication between mitochondria arose, in part, from their ability to
modulate propagation and integrity of Ca2+ waves under
physiological conditions (Jouaville et
al., 1995). Such notions were refined to encompass mitochondrial
Ca2+-dependent Ca2+ release mechanisms
(Ichas et al., 1997
) and
mitochondrial ROS-induced ROS release waves
(Zorov et al., 2000
). In
excitable cells such as myotubes, lateral signalling between mitochondria
induced by exogenous apoptotic stimuli was shown to be propagated by cytosolic
Ca2+ waves. These Ca2+ waves in myotubes also induced
travelling mitochondrial waves that involved depolarisation by the
mitochondrial permeability transition (MPT) and release of cyt c, leading to
caspase activation and nuclear damage characteristic of apoptosis
(Pacher and Hajnóczky,
2001
).
In non-excitable cells, coordination between subsets of mitochondria
remains poorly explored in these aforementioned terms. Electrical coupling
between mitochondria under non-pathological conditions has been shown in COS-7
cells (De Giorgi et al., 2000)
and human fibroblasts (Amchenkova et al.,
1988
; Diaz et al.,
2000
). This coupling takes the form of
m
flickering in individual mitochondria or of synchronous depolarisation in
whole subsets of mitochondria, suggestive of intermitochondrial communication.
We recently demonstrated intermitochondrial communication in non-excitable
human osteosarcoma 143B TK- cells during apoptosis, by applying a
microscopic photosensitisation technique
(Lum et al., 2002
) with the
mitochondria-specific dye Chloromethyl-X-Rosamine (CMXRos; MitoTracker Red), a
potent photosensitiser (Minamikawa et al.,
1999a
). We have used the laser scanning beam of a confocal
microscope to irradiate a subset of mitochondria in an individual cell. This
resulted in complete depolarisation of non-irradiated mitochondria in the same
cell (Lum et al., 2002
). In
the present study we have used this system to distinguish two temporally
distinct phases of signalling between irradiated mitochondria and their
non-irradiated counterparts, namely the initial rapid depolarisation of the
non-irradiated organelles and their subsequent delayed release of cyt c. These
findings extend the current knowledge of intermitochondrial communication and
show that multiple modes of such interorganellar signalling occur in stressed
cells undergoing apoptosis.
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Materials and Methods |
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Cell lines
Human osteosarcoma 143B TK (hereinafter called 143B)
cells were cultured as previously described
(Minamikawa et al., 1999b).
For generation of 143B cells stably overexpressing Bcl-2, cells were
transfected with pEF Bcl-2 pGKpuro (gift from D. Huang, Walter and Eliza Hall
Institute, Melbourne) using Lipofectamine (Invitrogen). Transfectants were
selected by growth in puromycin (50 µg/ml). Cell lines were generated from
single cells cloned using limiting dilution culture. Clones expressing high
levels of Bcl-2 were identified by immunofluorescence staining of
permeabilised, fixed cells with monoclonal anti-Bcl-2 antibody (Bcl-2-100;
Sigma). One such clone was selected and used throughout this study. The cell
line expressing GFP targeted to the mitochondrial matrix (MtGFP) was
constructed by stably transfecting 143B cells with pCZ34
(Zhang et al., 1998
) as
previously described (Lim et al.,
2001
).
143B cells stably expressing cyt c-GFP were generated by infection of cells
with retroviral supernatant (kindly provided by J. Goldstein, La Jolla
Institute for Allergy and Immunology, San Diego, CA) containing the vector
pBabe-puro with the cloned insert being mouse cyt c tagged at its C-terminus
with GFP. Cells were infected at 50% confluency and two rounds of
infection at 37°C were carried out at a ratio of 1:2 (v/v) viral
supernatant to cells. The duration of each round of infection was 6-8 hours.
Cells were then left to grow in fresh media until confluent prior to
subculturing into 96-well plates for obtaining single cell colonies by
limiting dilution. Clones expressing cyt c-GFP were selected in puromycin (0.5
µg/ml) and screened by confocal analysis of cyt c-GFP fluorescence and
localisation to mitochondria. In each clone so retrieved, a mixed population
was encountered that consisted of cells with varying levels of expression and
cyt c-GFP localisation.
However, for the purposes of the microscopic photosensitisation studies in this work, only cells that show expression of cyt c-GFP fluorescence predominantly in mitochondria were irradiated.
Microscopic photosensitisation by partial irradiation
Cells were seeded the day before irradiation into 35 mm dishes at a density
of 6x104 cells per dish, the bottom of which was fenestrated
and sealed with a round grid-coverslip (CELLocate, Eppendorf, grid size 175
µm). For photosensitisation, cells were loaded with CMXRos (200 nM, 15
minutes at 37°C) and washed with phenol-red-free RPMI 1640 medium (Gibco
BRL Life Technologies). Cells were maintained at 37°C in a
temperature-controlled chamber (Life Science Resources, Cambridge, UK) on the
stage of an inverted Leica DMIRB confocal microscope (TCS-NT system). Single
cells targeted for photoirradiation were initially observed under dim
transmission illumination from a tungsten lamp and the cellular position on
the grid coverslip noted. The target cell was then positioned at the centre of
the visual field and an image of the whole cell was obtained at low laser
intensity (zoom 1, 2-4 scans) to minimise phototoxicity. A portion of the cell
(typically half of the cell) was photoirradiated at high laser intensity to
induce damage on a subpopulation of mitochondria in a given cell. Typically,
partial irradiation was conducted by continuous xy scanning using the 488 and
568 nm excitation from the Ar/Kr laser at 128 scans (1 second/scan), zoom 8,
and imaged with a Leica PL APO 63x/NA 1.2 water immersion objective.
Immediately after irradiation, the whole cell was imaged at low laser
intensity (zoom 1, 2 scans) and the same cell monitored at appropriate time
points following subsequent staining with other fluorescent indicators for
cell death events. Unstained cells that were partially irradiated under these
conditions were able to undergo cell division more than 8 hours after
irradiation.
Imaging
Fluorescent images were obtained under confocal microscopy conditions as
above. Excitation wavelength/detection filter settings for each of the
fluorescent indicators were as follows: CMXRos and propidium iodide (PI),
568/665-nm longpass; GFP, Alexa Fluor-488, Fluo-3 and dichlorofluorescein
(DCF), 488/530-nm bandpass filter. For simultaneous imaging of CMXRos with
either Rh123, GFP, Alexa Fluor-488, Fluo-3 or DCF, laser excitation used was
488 and 568 nm. Image analysis and processing was performed with Image J
(National Institutes of Health, USA).
Assessment of m
Most experiments used Rh123 (10 µM) loaded into cells for 15 minutes at
37°C. In some experiments, TMRM (150 nM) was used instead of Rh123 and
cells were incubated with TMRM for 20 minutes at 37°C subsequent to
partial irradiation. Following loading with either Rh123 or TMRM, cells were
washed and maintained in phenol-red-free RPMI 1640 medium (also containing 50
nM TMRM for cells stained with TMRM) for confocal imaging. In the case of
Rh123 loading, cells were scored as manifesting high m
or loss of
m, respectively, in their non-irradiated
mitochondria on the basis of Rh123 retention values determined previously
(Lum et al., 2002
) that were
either above 60% or below 20% of controls.
Dismemberment of mitochondrial filamentous networks
To induce conversion of filamentous mitochondria into discrete punctate
entities, cells were treated with CCCP (20 µM) for 60 minutes followed by
washout of CCCP with fresh RPMI 1640 media. After incubation for 60 minutes at
37°C, cells had recovered their m
(Minamikawa et al., 1999b
) and
were then loaded with CMXRos prior to irradiation.
Assessment of cell death pathway indications
Release of cytochrome c into the cytosol and translocation of Bax to the
mitochondria, respectively, were assessed by immunocytochemical staining of
cyt c (Lum et al., 2002) and
Bax (Lim et al., 2001
). Cyt c
release from mitochondria was indicated by weaker diffuse staining across
whole cell sections that did not colocalise with mitochondria.
Phosphatidylserine (PS) exposure on the surface of the plasma membrane was
detected by an annexin V binding assay as described previously
(Lum et al., 2002
). Cells that
showed annexin V binding typically displayed a fluorescent ring on the
periphery of the cell. Uptake of propidium iodide (PI) was assessed by
incubating cells as described (Lum et al.,
2002
); necrotic (but not apoptotic) cells showed staining of
nuclei contingent on plasma membrane permeabilisation. Cells were imaged by
confocal microscopy for these various parameters relating to activation of the
apoptotic cell death pathway.
Measurement of intracellular Ca2+ and ROS levels
For detection of intracellular Ca2+ levels, cells were loaded
with the membrane-permeant Fluo-3 AM (2.5 µM) and incubated for 10 minutes
at 37°C followed by washout of excess dye with phenol red-free media. To
detect intracellular ROS levels, cells were loaded with the membrane-permeant
H2DCFDA following partial irradiation. Staining of cells with
H2DCFDA (40 µM) was performed in serum-free media by incubation
for 20 minutes at 37°C followed by washout of excess dye with phenol
red-free media. H2DCFDA is hydrolysed by intracellular esterases to
form DCFH (the reduced form of DCF) upon cleavage of the acetate groups on
H2DCFDA (Sawada et al.,
1996). ROS such as H2O2 and hydroxyl radical
but not superoxide, readily oxidise DCFH to result in the fluorescent DCF
(Vanden Hoek et al., 1997
).
The intracellular Ca2+ and ROS levels in each partially irradiated
cell were quantified using Image J by measuring the mean Fluo-3 and DCF
fluorescence, respectively, in each irradiated cell in pixel units expressed
relative to fluorescence of non-irradiated cells in the same field.
Statistical analysis
The cellular responses to partial irradiation (including loss or retention
of m, cyt c release, cyt c-GFP release, PS exposure,
and PI uptake) were scored as a percentage of the total cells analysed in each
category. To assess whether the responses between two different treatments
were significantly different (P
0.05), the relevant proportions of
cells from each treatment were comparatively analysed by a chi-squared
contingency test using Microsoft Excel 2000 software. A student's unpaired
t-test was used for assessing any significant difference in the mean
fluorescence value of Fluo-3 and DCF, comparing two groups of cells subjected
to different treatments.
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Results |
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In cells not exposed to CCCP, if one filament was a single long
mitochondrion which allows relay of energy (represented by
m) along the whole filament to facilitate
intramitochondrial communication
(Amchenkova et al., 1988
), then
one would expect a less efficient response to partial irradiation when the
filaments are dispersed by CCCP into smaller separate entities. However, our
data show that both the
m loss
(Fig. 2A) and the death
responses (Fig. 2B) in cells
containing discrete punctate mitochondria were not significantly different
(P>0.1) from those containing filamentous mitochondria. Moreover,
filamentous mitochondria in CMXRos-loaded cells that were subjected to
irradiation of 128 seconds themselves showed fragmentation into punctate
structures by 60 seconds (Fig.
3A), hence preventing signal diffusion along the whole filament
within the irradiated region. The high laser intensity utilised under such
partial irradiation conditions did not result in the fragmentation of
filamentous mitochondria in control cells
(Fig. 3B) not loaded with
CMXRos (but labelled with mitochondrial matrix-targeted GFP). Taken together,
these data suggest that
m loss in non-irradiated
mitochondria occurs via intermitochondrial and not intramitochondrial
communication.
|
Response generated in non-irradiated mitochondria is not mediated by
caspase-dependent amplification of signal from irradiated mitochondria
It has been proposed that caspases and mitochondria can engage in a
self-amplification loop in which the release of mitochondrial apoptogenic
proteins activates caspases that would in turn increase the mitochondrial
membrane permeability (Marzo et al.,
1998). Additional episodes of cyt c release may occur, which have
been shown to be due to caspase-3-dependent cleavage of Bcl-2
(Kirsch et al., 1999
;
Chen et al., 2000
). To
determine whether the signal originating from irradiated mitochondria is
amplified by downstream caspases, we treated cells with a broad spectrum
caspase inhibitor zVAD-fmk (100 µM) prior to partial irradiation. zVAD-fmk
at this concentration was effective in preventing caspase-3 activation and
cell killing following apoptotic induction by STS and MT-21 in 143B cells
(data not shown). However, zVAD-fmk did not inhibit either
m loss in non-irradiated mitochondria or cyt c release
(Table 1). This suggests that
caspases are involved neither in depolarisation of non-irradiated mitochondria
nor in cyt c release that is known to occur upstream of caspase-3
activation.
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Cyt c is released from both non-irradiated and irradiated
mitochondria during intermitochondrial signalling
Our previous study using immunocytochemistry revealed the release of cyt c
into the cytoplasm following partial irradiation
(Lum et al., 2002). However,
it was not possible to identify the source of such dispersed cyt c (either
from irradiated or non-irradiated mitochondria) because cells are fixed at a
certain point in time and the dynamics of cyt c release events could not be
readily analysed. To resolve whether non-irradiated mitochondria are activated
by irradiated mitochondria to release cyt c during the initiation of
apoptosis, we employed 143B cells that stably express cyt c-GFP targeted to
mitochondria. These cells behave similarly to the parent cell line in response
to apoptotic inductions by STS (0.5 µM, 1 µM),
H2O2 (1 µM, 2 µM), and MT-21 (50 µM). This was
revealed by the lack of significant difference between the two cell lines in
terms of cell viability and proportion of cells showing cyt c release
following the various apoptotic inductions (data not shown). We also
ascertained that in general the cyt c-GFP reporter follows bulk cyt c
localisation by performing immunocytochemical staining with cyt c antibody on
cyt c-GFP expressing cells subjected to the various apoptotic inducers above,
including treatment with STS, H2O2 and MT-21 (data not
shown). In the experiments below we use cyt c-GFP as a reporter of release of
cyt c from non-irradiated mitochondria following partial irradiation. For
technical reasons outlined below, the bulk cyt c release detected
immunochemically and the cyt c-GFP-derived fluorescence signal are now
dissociated.
Cells expressing cyt c-GFP, initially showing appropriate mitochondrial localisation of fluorescence, were subjected to partial irradiation after loading with CMXRos (Fig. 4A). This typically results in conversion of the bright punctate cyt c-GFP fluorescence in irradiated mitochondria into a diffuse, weak signal in the cytosol. This dispersal and loss of signal intensity results from a combination of the release of cyt c-GFP from irradiated mitochondria together with some extent of photobleaching of cyt c-GFP signal (green channel) in the irradiated mitochondria. The unbleached non-irradiated mitochondria, however, retain punctate mitochondrially localised cyt c-GFP fluorescence (Fig. 4A, 0 minutes). Therefore, it becomes possible to monitor subsequent cyt c-GFP release from the non-irradiated mitochondria. In the cell depicted in Fig. 4A, release is judged to be occurring about 25 minutes after partial irradiation and is complete after 30 minutes. Staining of non-irradiated mitochondria with CMXRos (red channel) remains clearly evident although a general reorganisation of mitochondria to cluster around the nucleus takes place in such apoptosing cells (Fig. 4A). Based on this principle, we detected cyt c-GFP release from non-irradiated mitochondria of 55% of cells tested within 2.5 hours subsequent to partial irradiation (quantified in more detail, below). Non-irradiated control cells in the same field do not release cyt c-GFP, as indicated by the retention of a substantial cyt c-GFP signal localised to mitochondria (Fig. 4B).
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To determine whether the cyt c-GFP release from the non-irradiated mitochondria is caspase-dependent, the cells were treated with zVAD-fmk (100 µM) before partial irradiation. A similar distribution of cells showing cyt c-GFP release at the various time intervals was obtained either in the presence (Fig. 5, open bars) or absence of zVAD-fmk (filled bars). Moreover, the proportion of cells that did not release cyt c-GFP by 2.5 hours remained unchanged by zVAD-fmk treatment (Fig. 5). These data suggest that the release of cyt c-GFP from non-irradiated mitochondria is not mediated by a caspase-dependent feedback amplification loop.
It is instructive to compare the timing of cyt c-GFP release from
non-irradiated mitochondria (delayed) with that of release of cyt c from
irradiated mitochondria which is already recognised to be a very early event
(within 30 minutes) for cells globally irradiated
(Minamikawa et al., 1999a).
Note that our partial irradiation technique emphasises fluorescence due to cyt
c-GFP within the non-irradiated mitochondria, while immunocytochemical
staining reveals the distribution of all cyt c within the cell. In our
experiments, the extensive release of cyt c detected by immunocytochemistry
occurs in the vast majority of cells by two hours after irradiation (88%,
n=27) [(Lum et al.,
2002
) and other data not shown]. In principle, this release
(measured in untransfected 143B cells) could have arisen from either
irradiated or non-irradiated mitochondria. However, as we have shown above,
using cells expressing cyt c-GFP, only about half (55%, n=33) have
released cyt c-GFP from non-irradiated mitochondria by 2.5 hours. We interpret
the difference in release between cyt c and cyt c-GFP to indicate that in most
partially irradiated cells, the immunocytochemically detectable early cyt c
release arises mainly from irradiated mitochondria. We also suggest that this
early cyt c release from irradiated mitochondria plays a major role in
activating the cell death pathway in response to CMXRos photosensitisation
(cf. Minamikawa et al.,
1999a
).
Depolarisation precedes the delayed cyt c-GFP release from
non-irradiated mitochondria
We next examined the relationship between cyt c-GFP release and
m loss following partial irradiation. Individual cyt
c-GFP expressing cells were monitored simultaneously for both cyt c-GFP
release and depolarisation which was detected by TMRM loaded after partial
irradiation with CMXRos. Note that TMRM and GFP fluorescence emission can be
detected in separate red and green channels respectively (whereas Rh123 and
GFP emissions overlap in the green channel). Even though the emission of TMRM
fluorescence is detected in the same red channel as CMXRos and is thus
recorded concurrently with the CMXRos fluorescence, it is still possible to
monitor
m based on uptake of TMRM in the mitochondria.
This is because CMXRos undergoes substantial photobleaching following
irradiation and any mitochondria that retain high
m do
show a restored red fluorescence upon subsequent loading of these
partially-irradiated cells with TMRM. The cyt c-GFP expressing cells analysed
in this manner showed that 88% of cells (n=16) underwent rapid
depolarisation in non-irradiated mitochondria consistent with that observed in
parent cells. Of these 16 cells, only 2 did not undergo such depolarisation,
their non-irradiated mitochondria becoming relatively brightly labelled with
TMRM (data not shown). In general, about half of cells which had undergone
depolarisation (9 out of 16) showed cyt c-GFP release from non-irradiated
mitochondria within 2 hours, the other 7 cells still retaining cyt c-GFP in
mitochondria at that time (consistent with data in
Fig. 5). These data indicate
that cyt c-GFP release is not an obligatory immediate consequence of
depolarisation in non-irradiated mitochondria.
Signalling is regulated by Bcl-2 but does not involve Bax and is not
inhibited by CsA
A hallmark of the participation of mitochondria in apoptotic signalling is
the regulation afforded by pro-apoptotic and pro-survival members of the Bcl-2
family (Desagher and Martinou,
2000). Overexpression of Bcl-2 in 143B cells was able to protect
against oxidative stress induced by hydrogen peroxide (data not shown), in
agreement with its ability to reduce the formation of ROS and prevent lipid
peroxidation mediated by oxygen radical
(Degli-Esposti et al., 1999
;
Hockenbery et al., 1993
).
Following partial irradiation of such Bcl-2 overexpressing cells, both
m dissipation in non-irradiated mitochondria and
release of cyt c were minimised (Table
1). This finding is consistent with the anti-apoptotic property of
Bcl-2 in stabilising the barrier function of mitochondrial membranes, for
example preventing cyt c release and interfering with loss of
m (Desagher and
Martinou, 2000
). Overexpressed Bcl-2 however did not markedly
protect globally irradiated cells from
m loss. All of
12 cells analysed under our standard intensive irradiation conditions showed
depolarisation in mitochondria throughout the cell, indicated by the failure
of any mitochondria to stain with TMRM loaded after irradiation (although we
have not systematically tried to find conditions of global irradiation that
might discriminate between control 143B cells and those overexpressing
Bcl-2).
By contrast, pro-apoptotic Bax is well known as a mediator of death
signalling, following translocation of Bax to mitochondria, which results in
cyt c release (Desagher and Martinou,
2000). We therefore used immunocytochemistry to examine whether
Bax is recruited to non-irradiated mitochondria in response to partial
irradiation. CMXRos was used as a mitochondrial marker in this study. In
contrast to the positive control treatment which showed mitochondrial
localisation of Bax in the great majority of cells following apoptotic
induction by STS, Bax was evidently not localised to the non-irradiated
mitochondria in any but a small minority of cases following partial
irradiation of CMXRos-loaded cells (Fig.
6). The loss of
m in non-irradiated
mitochondria is therefore not contingent on recruitment of observable
quantities of Bax from other cellular locations.
|
Finally, we tested whether CsA is able to block depolarisation of non-irradiated mitochondria. We found this not to be the case for CsA at the relatively high concentration of 20 µM (Table 1), nor at lower concentrations (data not shown). The significance of these findings in relation to a general model for the two phases of intermitochondrial signalling will be considered in the Discussion section.
Increased levels of intracellular Ca2+ mediate signalling
between mitochondria
We observed a significant increase in intracellular Ca2+ in
partially irradiated cells, as detected by Fluo-3 fluorescence (3-6 fold
greater than in non-irradiated cells in the same field
(Fig. 7A). Partially irradiated
cells typically show a brighter and diffuse Fluo-3 fluorescence in the entire
cell (Fig. 7B), distinct from
the weaker Fluo-3 signal in non-irradiated cells. The increased intracellular
Ca2+ levels following partial irradiation were reduced by
pre-treatment of cells with intracellular Ca2+ chelator BAPTA-AM
(Fig. 7A). Such reduction in
Ca2+ levels by BAPTA alleviated the loss of
m in non-irradiated mitochondria (43% of cells,
Table 1) and the occurrence of
cyt c release (41% of cells, Table
1), suggesting that increases in intracellular Ca2+
promote intermitochondrial signalling. Similarly, a significantly higher
intracellular ROS level measured by DCF fluorescence (4-8 fold greater than in
non-irradiated cells, Fig. 7C),
was also observed as a diffuse and bright fluorescence in partially irradiated
cells (Fig. 7D). Laser
irradiation on its own did not induce a cellular response as demonstrated by
minimal levels of either Fluo-3 fluorescence
(Fig. 7A) or DCF fluorescence
(Fig. 7B) following partial
irradiation of control cells not loaded with CMXRos.
|
Pretreatment of cells with NAC, an antioxidant that is also a metabolic
precursor to glutathione (GSH), also inhibited both m
loss in non-irradiated mitochondria and cyt c release to a similar extent as
BAPTA-AM (Table 1). However,
these latter effects of NAC could not be attributed to its function as an
antioxidant since it was unable to reduce intracellular ROS levels
(Fig. 7C); note that the DCF
signal was inhibited by catalase, a direct H2O2
scavenger (data not shown). It is possible that NAC could instead exert a
protective effect via its ability to restore intracellular GSH levels that are
depleted during apoptosis and thus prevent onset of MPT
(Liu et al., 2001
) and
ameliorate cyt c release. Interestingly, BAPTA-AM substantially inhibited the
increase in intracellular ROS levels (Fig.
7C) in partially irradiated cells loaded with CMXRos. This
suggests that the ROS increase may be mediated by the increased
Ca2+. Finally we showed that influx of extracellular
Ca2+ provides a major component of the enhanced intracellular
Ca2+ levels following photosensitisation of CMXRos-loaded
mitochondria (noting the reduced Fluo-3 fluorescence in cells photosensitised
in Ca2+-free medium; data not shown).
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Discussion |
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On the basis of data obtained here applying microscopic photosensitisation
by partial irradiation, we present a schematic that outlines our present
understanding of the intermitochondrial communication
(Fig. 8). This scheme indicates
how severe mitochondrial stress induced by CMXRos photoexcitation triggers
biphasic responses in non-irradiated mitochondria leading to apoptosis. Thus,
we have established that signalling between mitochondria during apoptosis
occurs in two temporally independent phases, notably early depolarisation
(induced within 10 minutes after irradiation) and followed by later cyt c
release that occurs at a delayed but unpredictable time after
m loss. Moreover, we suggest that Ca2+ is
likely to mediate the signal between irradiated and non-irradiated
mitochondria and that the elevated levels of intracellular Ca2+ may
also facilitate increases in intracellular ROS. The photodynamic aspects of
CMXRos photosensitisation themselves generate a burst of ROS, but we observed
that overall cellular ROS levels are suppressed by the Ca2+
chelator BAPTA. In other work, Ca2+ has been shown to increase ROS
production by disrupting the lipid organisation of the inner mitochondrial
membrane and, consequently the respiratory chain function
(Grijalba et al., 1999
). The
ROS produced induces permeabilisation of the inner mitochondrial membrane in a
non-specific manner. Moreover, mitochondrial ROS production during cell death
has been shown to be regulated by extracellular Ca2+ flux
(Tan et al., 1998
), consistent
with our observation that elevated intracellular Ca2+ in the
irradiated cells has a large extracellular Ca2+ derivation.
|
First phase of intermitochondrial signalling: early depolarisation in
non-irradiated mitochondria
We found that the early m loss in non-irradiated
mitochondria was not due to lumenal continuity of mitochondrial filaments in
the 143B cells used in our system. The lumenal discontinuity of mitochondrial
filaments has been reported in other cell types such as HeLa, Cos-7, cortical
astrocytes, cortical neuron, and human umbilical venous epithelial cells. It
is thought that such discontinuity facilitates the heterogeneous functioning
of mitochondria in relation to
m, sequestration of
Ca2+ and activation of MPT
(Collins et al., 2002
).
Propagation of
m along the whole filament
(Skulachev, 2001
) in our case
would be unlikely since mitochondria in the irradiated zone become physically
separated from their non-irradiated counterparts by fragmentation into smaller
structures (Fig. 3A). Such
fragmentation of irradiated mitochondria favours the model where mitochondrial
filaments represent multiple mitochondria connected by intermitochondrial
junctions (Skulachev, 2001
).
Indeed, this has been shown in 143B cells where each of the small mitochondria
is equipped with most or all of the metabolic and biosynthetic functions
(Margineantu et al., 2002
).
Disintegration of such filaments into smaller punctate mitochondria has also
been observed in cells treated with apoptotic inducers
(Frank et al., 2001
).
Pretreatment of cells with zVAD-fmk failed to abolish the early
depolarisation in non-irradiated mitochondria
(Table 1), indicating the lack
of participation of caspases in amplification of signal from irradiated
mitochondria. This supports a direct mode of signalling between mitochondria,
rather than secondary responses mediated by downstream feedback amplification.
A similar lack of involvement of caspases in intermitochondrial signalling in
myotubes was also noted (Pacher and
Hajnóczky, 2001). It is possible that some signal either
acting upstream or on the mitochondrial level mediates the rapid
depolarisation in non-irradiated mitochondria since the latter is inhibited by
overexpressed Bcl-2. In this respect, we have ascertained that Bcl-2 impeded
the non-irradiated mitochondria in responding to the signal from irradiated
mitochondria, rather than acting on the irradiated mitochondria to prevent
initiation of signal. This is based on our finding that global irradiation on
Bcl-2 overexpressing cells rapidly depolarised all mitochondria.
Possible nature of mitochondrial depolarisation events
To determine whether MPT is involved in mediating the
m loss in non-irradiated mitochondria following partial
irradiation, we pretreated the cells with the MPT inhibitor CsA prior to
irradiation. We found that depolarisation in non-irradiated mitochondria and
cyt c release were not inhibited, perhaps suggesting that MPT has not
occurred. However, selective inner membrane permeabilisation does occur after
global irradiation of CMXRos-loaded cells
(Minamikawa et al., 1999a
),
detected by the calcein-cobalt quenching procedure which was developed as a
reliable way of assessing MPT opening in living cells
(Petronilli et al., 1999
). Yet
in these globally irradiated cells, CsA did not block calcein release
(Minamikawa et al., 1999a
) in
contrast to cells undergoing MPT induced by CCCP
(Minamikawa et al., 1999b
).
Such insensitivity to CsA in partially irradiated cells (as well as in cells
subjected to global irradiation) could be attributed to the exposure of
mitochondria (possibly including non-irradiated mitochondria) to levels of
Ca2+ and ROS severe enough to render the MPT pore inhibitory
actions of CsA ineffective. In agreement with this proposition, Brustovetsky
and Dubinsky showed that in brain mitochondria which have been severely
depolarised by Ca2+ could not be repolarised by CsA
(Brustovetsky and Dubinsky,
2000
). Moreover, ROS may increase sensitivity of MPT to
Ca2+ through oxidation of thiol groups on the MPT pore, such that
opening occurs at high Ca2+ levels in a CsA-insensitive manner
(Halestrap et al., 1997
). We
therefore cannot eliminate the possibility that in the cases of both
irradiated and non-irradiated mitochondria the primary event of depolarisation
involves the opening of the MPT pore, but in a CsA-insensitive manner.
Second phase of intermitochondrial signalling: delayed cyt c-GFP
release from non-irradiated mitochondria
By employing a cell line that stably expresses cyt c-GFP, we found that cyt
c-GFP released from non-irradiated mitochondria was delayed, occurring at a
specifically unpredictable time but being rapidly completed (within 10
minutes) subsequent to the onset of release. The heterogeneous nature of the
onset of cyt c-GFP release (Fig.
5) is in general, similar to that described by Goldstein et al.
(Goldstein et al., 2000),
although in the latter case cyt c-GFP release is delayed even more than under
our partial irradiation conditions. Moreover, in this other study on apoptotic
induction not involving photosensitisation, mitochondria maintain
m (or may even be hyperpolarised) prior to release of
cyt c-GFP (Goldstein et al.,
2000
). But in the case of the partial irradiation studies we
carried out, mitochondria in almost all cases are clearly depolarised long
before cyt c-GFP release. This confirms that the release of cyt c from
mitochondria is independent of the state of polarisation of mitochondria and
cannot be obligatorily linked to MPT events
(Lim et al., 2001
), unlike in
some apoptotic systems where MPT and cyt c release seem to be coincident and
mutually interdependent (Kroemer et al.,
1998
).
We have found that cyt c-GFP release from non-irradiated mitochondria is
not a secondary event due to downstream caspase mediated amplification loop
(Fig. 5). This
caspase-independent release of cyt c-GFP is also observed in excitable
myotubes during intermitochondrial communication
(Pacher and Hajnóczky,
2001). Based on the data shown here, we suggest that
Ca2+ is involved in what appears to be direct intermitochondrial
communication.
Is Bax involved in cyt c release?
In an attempt to uncover the events leading to cyt c release from the
non-irradiated mitochondria, we looked for the participation of Bax. However,
we found no evidence of mass recruitment of Bax to mitochondria. In our
situation, we cannot exclude that Bax is recruited to non-irradiated
mitochondria, in amounts below the level of detection, which may trigger cyt c
release. The reduced Bax requirement may arise in circumstances where
relatively little Bax is needed because of previous chronic bombardment of
non-irradiated mitochondria with Ca2+ or other signals originating
from irradiated mitochondria, such as ROS. By contrast, in cells undergoing
STS-induced apoptosis the recruitment of Bax to mitochondria was the same
irrespective of whether mitochondria were polarised or not (the latter induced
by prior CCCP treatment) (Lim et al.,
2001). Unlike in other systems, where it has been reported that
MPT events serve as the signal for recruitment of Bax to mitochondria
(Smaili et al., 2001
;
De Giorgi et al., 2002
), the
depolarisation of mitochondria resulting from CMXRos photosensitisation does
not evidently lead to such Bax recruitment.
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Acknowledgments |
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