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Morphology of mitochondrial permeability transitionMorphometric volumetry in apoptotic cells.

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THE ANATOMICAL RECORD PART A 281A:1337–1351 (2004)
Morphology of Mitochondrial
Permeability Transition:
Morphometric Volumetry in
Apoptotic Cells
ANTONIO SESSO,1* MÁRCIA M. MARQUES,2 MARIA M.T. MONTEIRO,3
ROBERT I. SCHUMACHER,4 ALISON COLQUHOUN,5 JOSÉ BELIZÁRIO,6
SÉRGIO N. KONNO,1 TAHIS B. FELIX,1 LUIS A.A. BOTELHO,1
VANESSA Z.C. SANTOS,1 GUILHERME R. DA SILVA,7
MARIA DE L. HIGUCHI,8 AND JOYCE T. KAWAKAMI1
1
Laboratory of Immunopathology, Institute of Tropical Medicine, University of São
Paulo, São Paulo, Brazil
2
Department of Operative Dentistry, School of Dentistry, University of São Paulo,
São Paulo, Brazil
3
Department of Pathology, Faculty of Medicine, University of São Paulo, São Paulo, Brazil
4
Department of Biochemistry, Chemical Institute, University of São Paulo, São Paulo, Brazil
5
Department of Histology, Biomedical Institute, University of São Paulo, São Paulo, Brazil
6
Department of Pharmacology, Biomedical Institute, University of São Paulo,
São Paulo, Brazil
7
Department of Preventive Medicine, Faculty of Medicine, University of São Paulo,
São Paulo, Brazil
8
Laboratory of Pathological Anatomy, Hearth Institute, University of São Paulo,
São Paulo, Brazil
ABSTRACT
Here we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose
inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrial
membranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating into
the cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported in
studies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types of
rat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa,
WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically.
Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in ␮m3
and the volume fraction of the cytoplasm occupied by mitochondria in cells with typical nuclear signs of apoptosis and also in
CUNS were evaluated. The volume of the mitochondria with ruptured membrane represents at least 69% (47–89%) of the total
mitochondrial volume of the apoptotic cells. Thus, a considerable fraction of the cellular mitochondrial mass is or was in the state
of permeability transition and probably involved in enhancement of the apoptotic program. In all samples, a fraction of the cells
with normal nuclei possessed mitochondria with breached outer membranes as described above. In these cells, MPT occurred
before the appearance of the typical nuclear phenotype of the apoptotic cells. © 2004 Wiley-Liss, Inc.
Key words: morphometry; mitochondria; apoptosis; rupture of the outer mitochondrial membrane;
mitochondrial permeability transition
The mitochondrial permeability transition (MPT) refers
to an increase in the permeability of the inner mitochondrial membrane caused by nonselective agents, in apoptotic and necrotic cells (Lemasters et al., 1998). This condition is associated with membrane depolarization and
collapse of the transmembrane potential (⌬⌿m). Due to
the hyperosmolarity of the matrix and the loss of selective
permeability of the inner membrane, the ensuing influx of
liquid to the mitochondrial matrix expands the matrix,
promoting a large swelling of the mitochondria. The loss of
selective permeability was initially thought to be caused
by defects in the lipid moiety of the membrane, consequent
©
2004 WILEY-LISS, INC.
Grant sponsor: Fundação de Amparo a Pesquisa do Estado de São
Paulo; Grant number: 00/06648-2; Grant sponsor: Conselho Nacional
de Pesquisas; Grant number: 520359/96-8; Grant sponsor: Pró-Reitoria
de Pesquisa da University of São Paulo (Procontes 2001).
*Correspondence to: Antonio Sesso, Instituto de Medicina Tropical de São Paulo, Av: Enéas de Carvalho Aguiar, 500 Prédio II 2°
andar, CEP 05403-000 São Paulo, SP, Brasil. Fax: 55-11-30667065. E-mail: antses88@uol.com.br
Received 3 March 2004; Accepted 20 July 2004
DOI 10.1002/ar.a.20134
Published online 5 November 2004 in Wiley InterScience
(www.interscience.wiley.com).
1338
SESSO ET AL.
2⫹
to activation of phospholipase A2 by elevated Ca levels
(Pfeiffer et al., 1979; Beatrice et al., 1980). The alternative
hypothesis, that MPT results from the opening of a pore or
megachannel, referred to as the permeability transition
pore (PTP) transversing both mitochondrial membranes
(Hunter and Haworth, 1979; Zoratti and Szabo, 1995;
Bernardi, 1999), has been extensively investigated and is
currently widely accepted.
It was thought that, after the onset of MPT, expansion
of the swollen matrix would cause the mitochondrial outer
membrane to rupture due to the limited capacity for distension of this membrane. The inner membrane may expand much more because of its continuity with the membrane of the cristae (Petit et al., 1998). The possibility that
the rupture of the outer membrane would allow the release of cytochrome c and other mitochondrial intermembrane proteins (Petit et al., 1998) is one of various alternative mechanisms found in the literature to explain how
these protein inducers of apoptosis reach the cytoplasm.
Another hypothesis is that the proteins are released to the
cytoplasm by passing through the outer membrane (Desagher and Martinou, 2000). The idea that the release of
mitochondrial proteins occurs either through permeabilization of (Basañez et al., 1999; Belzacq et al., 2002; Ravagnan et al., 2002) or the formation of supramolecular
openings in (Antonsson et al., 2001; Kuwana et al., 2002)
the outer membrane has recently gained momentum.
In in vitro systems, the proteins of the PTP may interact
with proapoptotic proteins, such as Bax and Bid, promoting the permeabilization of the outer mitochondrial membrane to cytochrome c (Zamzami and Kroemer, 2003).
MPT has been described in necrotic (Nieminen et al.,
1995, 1997; Kim et al., 2003) and the majority of apoptotic
cells. There have been reports of cases where the mitochondria of apoptosis-induced cells release cytochrome c
without exhibiting permeability transition (PT) (Vander
Heiden et al., 1997; Goldstein et al., 2000); however, this is
not clearly understood (Tafani et al., 2001). In the majority of reports (Belzacq et al., 2002; Castedo et al., 2002),
MPT is directly associated with the initiation of the apoptotic process.
Once in the cytoplasm, cytochrome c assembles with two
other proteins, the apoptotic protease-activating factor 1
(Apaf-1) and procaspase 9 to form a complex, the apoptosome. The ensuing activation of caspase 9 leads the cell to
the execution phase of apoptosis.
In addition to cytochrome c, other intermembrane mitochondrial proteins, such as Smac/DIABLO and Omi/
Htra2, are able to induce or enhance the activation of
caspases. The intermembrane proteins AIF and endonuclease G may act independently of caspase activation (Kuwana and Newmeyer, 2003). According to the cell type,
caspases 2, 3, and 9 may be added to the list of mitochondrial intermembrane proteins that enhance the apoptotic
process when they are liberated into the cytoplasm (Susin
et al., 1999a, 1999b).
When apoptosis is induced by the other major route of
induction, occupation of the TNF receptors, there are
many cases in which the mitochondrial pathway is responsible for the manifestation of apoptosis. This seems to
occur when relatively low levels of caspase 8 are activated
near the cytoplasmic portion of the occupied TNF receptor.
In this instance, procaspase 3 cannot be directly activated
by caspase 8 to implement the late stages of the apoptotic
program. However, caspase 8 can cleave the cytosolic pro-
tein Bid, giving rise to the truncated form of Bid, tBid,
which translocates to the outer mitochondrial membrane,
where it will interact with Bax and other proteins to
promote the release of the mitochondrial intermembrane
death-inducing proteins (Scaffidi et al., 1998; Antonsson
et al., 2001). These facts emphasize the extensive participation of mitochondria in the enhancement of the apoptotic program.
Examination using transmission electron microscope
(TEM) sections from various cell types undergoing programmed cell death identified many mitochondria with
ruptured outer membranes. Through the breach, the swollen mitochondrial matrix, covered by an expanding inner
membrane, herniates into the cytoplasm. This structural
change in the mitochondria is identical to that previously
described by Angermüller et al. (1998). The generalized
rupture of the outer mitochondrial membrane revealed a
wide range of microscopic profiles. Some of these profiles
resemble in vitro TEM experiments in which isolated mitochondria swelled to various degrees of induction of the
permeability transition. These results will be commented
on below. Based on these observations, it seemed of interest to evaluate the magnitude and latitude of mitochondria undergoing the permeability transition in apoptotic
cells from various cultured lineages induced with various
apoptogenic agents. We have obtained estimates of the
volume per cell and the cytoplasmic volume fraction of
mitochondria with ruptured and nonruptured outer membranes in apoptotic and companion nonapoptotic cultured
cells (CUNS). The cellular volume of mitochondria is the
product of the number of mitochondria per cytoplasm and
the average individual mitochondrial volume. Thus, by
analyzing how these parameters changed, we hope to obtain an indirect insight into how the number of mitochondria was affected by the early stages of apoptosis. In the
four cell lines studied morphometrically, we observed that
the volume of mitochondria with ruptured outer membrane in apoptotic cells represents from 47% to 89% of the
measured cell total mitochondrial volume. In all studied
samples, we noticed that a fraction of the CUNS also
possessed mitochondria with a breached outer membrane.
In these cells, MPT preceded the activation of the caspases
that induce the apoptotic nuclear phenotype. Although
these results do not prove that the rupture of the outer
mitochondrial membrane is the mechanism by which the
intermembrane proteins are released into the cytoplasm,
it is a most likely possibility.
MATERIALS AND METHODS
Electron Microscopy
The organ fragments and cultured cells were processed
as previously described (Sesso et al., 1999). Silver sections
stained with uranyl acetate and lead citrate (both from
Ladd Research Industries) were observed using a Jeol
1010 electron microscope or a Philips 301 at 80 kV. When
it was necessary, to check whether the outer mitochondrial membrane was actually in the section but could not
be observed due to an unfavorable sectioning angle, we
performed a 24° tilt on either side of the normal plane of
observation. This plane is at 0° tilt.
Rat Tissue Cells
Cells obtained from rat tissue were as follows: secretory
epithelial cells from the ventral lobe of the prostate gland
MITOCHONDRIAL PERMEABILITY TRANSITION
from rats killed daily in the 2–10 days following castration
(Kyprianou and Isaacs, 1988); plasma cells from the granulation tissue of an experimentally induced scar in the
dorsal skin of adult rats; macrophage, from the same
granulation tissue; pancreatic acinar cells from pancreatic
glands that had undergone ligature of the excretory ducts
(Gukovskaya et al., 1996) 2– 4 days previously or from rats
maintained on a protein-depletion diet and receiving daily
injections (40 mg/100 g of body weight) of dl-ethionine
(Sigma) for 5 days (Walker et al., 1993); and secretory
mammary cells from female rats subjected to interrupted
lactation with daily gland removal from days 1 to 10.
Cultured Cells
PC-12 cells were kindly supplied by Dr. Paulo Lee Ho
from the Institute Butantan in São Paulo. These cells
were derived from PC-12 cells obtained directly from
ATTC (pheochromocytoma; rat; ATCC CRL-172). They
were grown in Dulbecco’s modified Eagle’s medium
(DMEM; Life Technologies) supplemented with 10% FCS
(Cultilab) at 37°C in a humid atmosphere in 5% CO2. After
being stabilized in these growth conditions (Ho and Raw,
1992), it is no longer necessary to add poly-L-lysine to the
underlying support as the cells adhere more easily than
the original PC-12 cells. The adapted cells used here are
referred to as PC-12* cells. The PC-12* cells were serumdeprived for 3 and 8 hr or exposed to brefeldine A (BFA 2.0
␮M; Calbiochem; which blocks the anterograde vesicular
traffic from the ER to the Golgi, but not the retrograde
traffic), to 0.5 ␮M of staurosporine (STS; Calbiochem; a
protein kinase C inhibitor), and to BFA ⫹ STS in the same
concentrations, with an exposure time of 16 hr.
WEHI-3 cells (myelomonocyte leukemia; mouse; ATCC
TIB-68) maintained in RPMI-1640 (Sigma) plus 10% calf
serum were exposed for 5 hr to 20 ␮g/ml of the teneposide
VM 26 inhibitor of topoisomerase II; 0.4 ␮g/ml vimblastine
(Calbiochem; which prevents tubulin polymerization; 400
␮g/ml of the antibiotic novobiocin (Sigma); 0.5 nM okadaic
acid (Sigma), a potent inhibitor of protein phosphatases 1
and 2A; and 0.5 ␮M STS.
K-562 cells (chronic myelogenous leukemia; human;
ATCC (CCL-243) exposed for 5 hr to vimblastine 0.4 ␮g/
ml; oligomycin (Sigma), a highly specific mitochondrial
ATP-synthase inhibitor (5 nM); VM 26, 20 ␮g/ml; novobiocin (Sigma) 400 ␮g/ml; nigericin (Calbiochem), a protonionophore, 10 ␮M; and BFA 2.0 ␮M.
HeLa cells (epithelioid carcinoma; cervix; human; ATCC
CCL-2) in medium devoid of serum were exposed to 0.5
␮M STS plus 2.0 ␮M BFA or to 0.5 ␮M STS for 16 hr.
WEHI-164 cells (mouse; methylcholanthrene-induced
fibrosarcoma; ATCC CRL-1751) were exposed to BFA, to
STS, and to BFA ⫹ STS in the same conditions previously
employed.
HL-60 cells (human; peripheral blood; promyelocytic
leukemia; ATCC CCL-240) were exposed for 16 hr to BFA
2 ␮M ⫹ human TNF 100 ng/ml ⫹ 2 ␮M camptothecin
(CAMP; Sigma; this drug is a topoisomerase I drug that
acts primarily on cells in the S-phase of the proliferative
cycle). WEHI-3, K-562, and HeLa cells were cultured in
RPMI-1640 with 10% FBS (Cultilab) in a humid atmosphere at 37°C in 5% CO2.
The apoptogenic agents for all samples were added to
the cultures in fresh medium when the cells were about
50 – 60% confluent. In earlier phases of this study, we
examined cultures of WEHI-3, K-562, L 929 (mouse fibro-
1339
sarcoma; CLL-1.1), LLC-WRC 256 (carcinoma Walker;
rat; ATCC CCL-38), WEHI-164, HL-60, and PC-12 cells
(pheochromocytoma; rat; CRL-172). The PC-12 cells were
obtained from the American Type Culture Collection in
Rockville in 1992. Samples from all these cell lineages
were analyzed under TEM in the early stages of exponential growth and not exposed to apoptogenic drugs.
Morphometric Procedures
The morphometric study was carried out on the four
possible sectional profiles of mitochondria exhibiting rupture of the outer membrane (Fig. 1). Mitochondria with an
intact outer membrane in apoptotic and CUNS cells from
the same culture are referred to as type 1 mitochondrion
and their sections are named type 1 profiles (Fig. 1A). To
facilitate the identification of these profiles, we refer to the
mitochondrion with a breached outer membrane as a type
2 mitochondrion (Fig. 1B). When it is sectioned unequivocally, we refer to it as a type 2 mitochondrial profile
(schematized in Fig. 1D). A type 2 mitochondrion may be
sectioned in such a way as to create mitochondrial profiles
with both membranes (Fig. 1C) or only one membrane
(Fig. 1E and F). The vesicular profiles covered only by the
inner mitochondrial membrane are unequivocally recognized as type 3 profiles when they contain the remnants of
mitochondrial cristae (Fig. 1E). Mitochondria covered by
only the inner membrane and containing no cristae are
referred to as type 4 profiles (Fig. 1F). We measured only
profile types 1, 2, and 3. When a cell exhibits types 1 and
2 mitochondria, we cannot be sure from which of these two
types a given type 1 profile originates (Fig. 1C).
Estimates of the cytoplasmic volume and the volumes
associated with the mitochondrial profile types 1, 2, and 3
in apoptotic cells and the type 1 profile in nonapoptotic
cells. These parameters were obtained by point-counting
volumetry as indicated by Aherne and Dunnill (1982),
Sesso et al. (1999), and Gundersen et al. (1988).
The equivalence of area fraction (AA) and volume fraction (Vv) by which AA ⫽ Vv is a fundamental concept of all
morphometry (Aherne and Dunnill, 1982). Areas are estimated with a known degree of accuracy by counting how
many regularly spaced points of a test system fall inside
the area (hits). A given area value at the microscopic
magnification chosen is associated with each point. This
approach was used to estimate the areas of the cell sections from which we needed to obtain the radii. We will
briefly survey the complete morphometric procedures
used. The area fraction, AA, and, by extension, the volume
fraction, Vv, also referred to as volume density, occupied
by the mitochondria in the cytoplasm, is obtained by
counting hits over the mitochondrial profiles (be they 1, 2,
or 3) and over the cytoplasm including the previous counts
over the mitochondrial profiles. The ratio between these
two counts is AA ⫽ Vv. In order to obtain the cytoplasmic
volume, the percentage of the cell volume the cytoplasm
represents and its absolute value in ␮m3 must be calculated. To estimate the cellular volume, the distribution of
radii of the cell sections is obtained. The cytoplasmic volume fraction of the cell is obtained by counting hits over
the nuclear profiles and over the cytoplasm. The ratio
between the number of hits over the cytoplasm and over
the entire cell profile (the sum of hits over nuclei and
cytoplasm) is the parameter procured. The cellular volume
is obtained independently by measuring the radii of the
cell sections.
1340
SESSO ET AL.
Fig. 1. A schematic drawing illustrating how different angles of the
sectioning plane will furnish different profiles in normal type 1 mitochondrion (A) and mitochondrion with a ruptured outer membrane (type 2
mitochondrion; B and D). Type 1 profiles can be generated from type 1
or 2 mitochondrion as demonstrated in C. The generation of profile types
3 and 4 is demonstrated in E (containing cristae) and F (without cristae),
respectively. In all micrographs, the bars measure 500 nm.
The cellular and cytoplasmic volume of each cultured
cell type was estimated using measurements from 10 to 20
enlarged prints (2,000 ⫻ 2.5). Each recorded microscopic
field possessed 2– 8 profiles of CUNS and at least one
profile of a cell in explicit apoptosis. To be considered as a
cell undergoing apoptosis, in addition to the nuclear phenotype of apoptosis, it had to have most of its perimeter
covered by the cell membrane and no signs of advanced
proteolysis. Some 15–30 and 70 –100 profiles of apoptotic
and CUNS cells, respectively, were measured. The test
system superimposed over the prints was composed of
three types of hit marker, regularly spaced small crosses
and the extremities of two different types of regularly
spaced segments of straight lines (Gundersen et al., 1988).
Estimates of the volume density (Vv) of the nucleus and
cytoplasm of the cells were thus obtained [Vv of the cytoplasm (Vvc) ⫽ number of hits over the cytoplasm/number
of hits over the nucleus plus the number of hits over the
cytoplasm]. Since the cell sections are fairly circular, the
number of hits over each cell section gives an estimate of
the corresponding area (␲r2). In each sample, the radii
from apoptotic and cells with unaltered nuclear structure
were thus obtained. A 10-class distribution of radii was
constructed. Employing the procedure of Bach (1963),
used either in an HP machine (Arcon et al., 1980) or in a
PC, the main parameters, including the mean sphere volume of the corresponding distribution of radii, were obtained. Since the mean sphere volume is an estimate of
the cellular volume (vcell), the cytoplasmic volume is vcyt ⫽
vcell ⫻ Vvcyt.
AAmit of the mitochondria in the cytoplasm, one obtains
the total mitochondrial volume or area (vtm or Atm). Thus,
vtm or Atm ⫽ Vvmit (or AAmit) ⫻ vcyt. Since a randomly
sectioned type 2 mitochondrion furnishes profile types 1,
2, 3, and 4, of which only the initial three profiles were
measured, the sum of the obtained volumes for profile
types 2 and 3 gives the best possible estimate of the
volume of type 2 mitochondria. This volume, however, is
forcibly underestimated on two accounts. In apoptotic
cells, the type 1 profiles derived from type 2 mitochondria
cannot be scored as such. Likewise, all type 4 profiles are
not counted. Therefore, the mitochondrial volume derived
from evaluations carried out on type 1 profiles in apoptotic
cells represents the aggregated volume of actual type 1
and some type 2 mitochondria. The estimates of the cytoplasmic volume (or area) associated with mitochondrial
types 1, 2, and 3 profiles in apoptotic cells were carried out
using prints enlarged to 20,000⫻ (8,000 ⫻ 2.5). The cytoplasm from both CUNS and cells undergoing apoptosis
were examined for each type of cell culture.
At least 10 micrographs were obtained from each group.
In the apoptotic cells, we often found the three types of
mitochondrial profiles. In CUNS, particularly from cultures with high apoptotic indexes, in addition to profile
type 1, we also observed mitochondrial profile type 2
and/or 3. These mitochondrial profiles, except for the case
in line 2 of Table 1, were not point-counted as explained
above. In the enlarged (20,000⫻) micrograph, each type of
mitochondrial profile was marked with a fine ink dispenser. The number of hits falling within each profile type
(H1, H2, and H3, for mitochondrial profile types 1, 2, and
3, respectively) was scored. The sum of all mitochondrial
hits (H ⫽ H1 ⫹ H2 ⫹ H3) and the total number of hits (h)
over the cytoplasm was also scored (h ⫽ the sum of hits
over mitochondrial and nonmitochondrial cytoplasmic
structures). The volume density of the total mitochondrial
types in the cytoplasm (Vvtm) is Vvtm ⫽ H/h; and the Vv of
Volumes of Mitochondria Types 1 and 2
Again, the area fraction was estimated by the pointcounting procedure. The volume fraction is more commonly referred to as volume density (Vv). Having the
cytoplasmic volume vcyt and, for example, the Vvmit or the
1341
MITOCHONDRIAL PERMEABILITY TRANSITION
TABLE 1. Estimates of the volume density (Vv) and of the volume (v) of types 1 and 2 mitochondria in
apoptotic (AP) cells and in CUNS*
Volume
density (Vv)
and volume
(v) of type 1
mitochondrial
profiles in
CUNS
Vv and v of
type 1
mitochondrial
profiles in AP
cells
Vv, v, and %
of type 2
mitochondrial
profiles in AP
cellsa
Vv and v of
type 3
mitochondrial
profiles in AP
cells
3-sera-deprived
PC-12* cells
Vv (%) 10
v (␮m3) 28
2.3
6
2.3
6
HL-60, BFA ⫹
TNF␣ ⫹
CAMP/16 h
PC-12*, BFA 2
␮M/16 h
Vv (%) 7.9
v (␮m3) 47c
4.4
27
Vv (%) 5.7
v (␮m3) 38
1
6
Vv (%) 7
v (␮m3) 57
0.6
3
Vv (%) 7
v (␮m3) 162
2.4
60
Vv (%) 4.5
v (␮m3) 97
0.5
12
Vv (%) 9.2
v (␮m3) 224
1.4
32
Vv (%) 12
v (␮m3) 297
3.7
91
1.5
4.0
25%c,d
5.9
36
54%
4.6
28
70%
4
17.0
77%d
1.9
48
42%
2.7
40
71%
2.3
52
52%
4.7
115
54%
Cell type and
treatment
PC-12*, BFA ⫹
STS, 2 ␮M
and 0.5 ␮M/
16 h
WEHI-164,
BFA 2 ␮M/
16 hr
WEHI-164,
STS 0.5 ␮M/
16 hr
WEHI-164,
BFA ⫹ STS,
2 ␮M and
0.5 ␮M/16 h
HELA, BFA ⫹
STS, 2 ␮M
and 0.5 ␮M/
16 h
0.4
3
0.6
4
0.3
2
0.2
5
0.2
4
0.7
16
0.3
8
Vv, v, and % of
summed Types 2
and 3
mitochondrial
profiles in AP
cellsb
Vv and v of
the total
mitochondrial
profiles in AP
cells
3.8
10
63%b
6.3
39
59%
5.2
34
89%d
4.3
19
86%
2.1
53
47%d
2.8
44
79%
3
68
68%
5
123
57%
6
16
10.8
66
6.2
38
4.9
22
4.3
113
3.4
56
4.4
100
8.7
214
*In CUNS exempted for the data in line 2 and column 2, only type 1 mitochondrial profiles were sampled.
a
Percentage of the estimated volume in relation to the total mitochondrial volume in AP cells.
b
Percentage of the estimated volume in relation to the total mitochondrial volume in AP cells.
c
In this sample, the volumes of types 2 and 3 mitochondrial profiles were evaluated in CUNS and were 19 and 0.3 ␮m3,
respectively.
d
Extreme percentual values in the columns.
each mitochondria type in apoptotic cells is Vvi ⫽ Hi/h,
where i refers to mitochondrial profile types 1, 2, and 3
and ⫽ 1, 2, and 3. The respective total mitochondrial
volume (vtm) is vtm ⫽ Vvtm ⫻ vcyt and the volume associated with each mitochondrial profile type is vmi ⫽ Vvi ⫻
vcyt.
Total Mitochondrial Surface Area on a Per-Cell
Basis in Apoptotic Cells and CUNS: Evaluation
of the Average Surface-to-Volume Ratio of
Mitochondria
The hits (hi) made by the extremities of the regularly
spaced segments [of length (l) in ␮m at the given magnification] of the test system, in the interior of the mitochondrial profiles (1, 2, or 3 in apoptotic cells) and also over the
remaining cytoplasm, were scored. The number of times
the segments from the test system crossed (C) the limiting
membranes of the mitochondrial profiles (1, 2, or 3 in
apoptotic cells and 1 profiles in CUNS) WAS also detected.
The mitochondrial surface density (Sv) is Sv ⫽ 4C/l ⫻ hi,
where C and hi are the total number of times the segments
of the test system cross the mitochondrial borders and the
number of hits the segment extremities superimpose on
the whole cytoplasm, including the mitochondrial profiles.
Sv is the mitochondrial surface area in ␮m2 per ␮m3 of
cytoplasm. The total mitochondrial membrane surface
area per cell (TMMSA) is obtained by multiplying Sv by
vcyt. The average cellular mitochondrial surface-to-volume
ratio (s/v) is s/v ⫽ TMMSA/vtm.
Error Associated With Area Estimation
The coefficient of error associated with the estimation of
mitochondrial areas, circular or elliptical, was kept below
the 0.05 level and was determined as described by Gundersen and Jensen (1987).
Apoptotic Index
With magnifications of 2,000 ⫻ 10 and 5,000 ⫻ 10, at
least 50 –100 cells sections were randomly picked and the
percentage of apoptotic cells were evaluated. The characteristic nuclear alterations of the apoptotic cells are unmistakably recognized using TEM. The apoptotic cells
were selected for morphometric studies if they possessed
both a typical apoptotic nucleus and a cell membrane at
the cytoplasmic border. For the estimation of the apoptotic
indexes (AIs), all sections of cells with the nuclear phenotype of apoptosis were counted. Therefore, cells in various
1342
SESSO ET AL.
advanced stages of cytoplasmic proteolysis were also
counted.
RESULTS
Detection of Apoptotic Cells by Transmission
Electron Microscopy
Among the biochemical and structural changes observed in cells undergoing apoptosis, the most prominent
and an essential element in the identification of this type
of cell death are the structural alterations of the nucleus.
Our observations confirm that the various forms of apoptotic nuclear phenotype are represented in both rat tissue cells and in immortalized cultured cells. Nuclear contraction is coincident with hypercondensation of the
chromatin (i.e., exceeding that seen in normal heterochromatin) and the clumping of the chromatin into masses
that adhere along the nuclear membrane. Such masses
vary in appearance from large spherical-like (Fig. 2A) to
demilune-like conformations (Fig. 2B and C). The fragmentation of these large masses, which often occupy the
entire nuclear profile, into various smaller spheroid bodies
also occurs. This particular nuclear phenotype corresponds to what the former cytologists called karyorrhexis.
Additional phenotypes of apoptotic cells nuclei as well as
of mitochondria in both CUNS and apoptotic cells may be
examined at http://www.sebepa.cjb.net/; the password is
“sessoimt.”
Apoptotic Cells in Rat Tissues
The finding of mitochondrial profile types 2 or 3, or
both, reveals the presence of a type 2 mitochondrion. In
populations with an elevated percentage of apoptotic
cells, this finding is frequent. All apoptotic tissue cells
we examined possessed type 2 mitochondria (arrows in
Figs. 3 and 4B). The low-magnification image of the
apoptotic cell in Figure 3A reveals a common structural
alteration seen in both apoptotic cells and in CUNS
undergoing MPT in a less stretched cytoplasmic form.
We observe the absence of organelles in some sectors of
the cytoplasm (2 in Fig. 3A) and their clustering in
others (3 in Fig. 3A).
Rat prostate secretory cells. The lack of male hormones induces a marked involution of the prostrate gland
consequent to death of the secretory epithelial cells by
apoptosis. Ten days after castration, the gland mass is
reduced to some 15% of the original weight. We have
studied glands removed 4 – 6 days after orchiectomy more
thoroughly (Fig. 3A); these exhibit a high incidence of
apoptotic epithelial cells.
Plasma cell and macrophage. A distinct increase
in the number of cells at the granulation tissue occurs 4 – 6
days after scar formation. At day 5, we found more apoptotic plasma cells (Fig. 2B) and macrophages (Fig. 2C)
than at other time intervals. This is coincident with the
proliferation time of fibroblasts, plasma cells, and macrophage.
Pancreatic acinar cells. Both procedures used to
induce apoptosis were highly effective. After ligature of
the excretory ducts, the pancreas gland regions that
suffered a lack of flowing secretion with consequent
compression of the cells exhibited generalized apoptosis
of the acinar cells. In one of these glands, we observed
a nonapoptotic cell with a mitochondrion exhibiting
a ruptured outer membrane (arrow in Fig. 4A). In
the majority of apoptotic cells, mitochondria undergoing permeability transition could be recognized (Fig.
4B).
Mammary gland secretory epithelial cells. The
abrupt removal of the suction stimulus from the breast
promotes a hormonal imbalance that induces apoptosis of
the secretory cells (Walker et al., 1989) and consequent
involution of the mammary glands. The incidence of apoptosis in the secretory cells is revealed by the rapid increase of intraepithelial macrophage filled with apoptotic
bodies containing remnants of the secretory cell cytoplasm. The percentage of macrophage in the epithelium
rose by day 3 after the interruption of lactation and remained elevated until day 9. We found type 2 mitochondria in the apoptotic bodies inside epithelial cells from a
gland obtained at day 3 after separation of the littermates
from the nursing female.
Apoptotic Cultured Cells
Examining the various cell cultures maintained under normal conditions without the addition of apoptogenic agents, in no instance did we find type 2 mitochondria in the nonapoptotic cells. The L 929 and the PC-12
cells from ATCC were also examined after treatment
with apoptogenic agents. TNF␣ was added to cultures of
L 929 cells while apoptosis was induced in the PC-12
cells by sera removal. In these particular experiments,
no apoptosis was detected in the L 929 cells. To confirm
this negative finding, a thorough search for apoptosis as
well as type 2 mitochondria in CUNS was carried out
with no success. After sera removal from the PC-12
cells, samples were collected at various time intervals
between 2 and 48 hr. While the apoptotic cells exhibited
type 2 mitochondria in all time intervals, in no instance
did we find mitochondria with a ruptured outer membrane in CUNS. We have examined more than 600
CUNS profiles looking for type 2 mitochondria. The
sera-deprived PC-12 cells were the most thoroughly examined as we analyzed samples sectioned serially and
semiserially for other studies (Sesso et al., 1999). In the
modified PC-12* cells from the Butantan Institute, we
found mitochondria with a ruptured outer membrane in
CUNS and also in apoptotic cells in the very first samples deprived of serum. These observations will be presented and discussed in a follow-up study.
WEHI-3 and K-562 were among the first cell cultures
examined and subjected to various apoptogenic substances. We observed type 2 mitochondria in apoptotic
WEHI-3 cells exposed to STS, novobiocin, VM 26, and
vimblastin and in apoptotic K-562 cells treated with BFA,
VM 26, and thapsigargin. All dead cells from cultures
subjected to apoptogenic agents so far tested using morphometric studies possessed a cell membrane and nuclei
with the typical apoptotic phenotype.
It is common to observe mitochondria with spherical
profiles along with the normal elongated mitochondria in
CUNS. The incidence of these profiles was influenced by
the degree of cell death occurring in the cell culture. In the
experiments presented in Table 1, the percentage of cells
undergoing apoptosis varied from 6% to 66%. The higher
this apoptotic index, the more frequently one could find
CUNS with spherical mitochondria, many of which exhib-
MITOCHONDRIAL PERMEABILITY TRANSITION
Fig. 2. A: Apoptotic PC-12* cell treated with BFA (2 ␮M for 16 hr).
The compact mass of hypercondensed chromatin (1) appears detached
from the nuclear membrane (2), apparently leaving clear the region
where the nuclear lamina dwells (the small black dot at the extremity of
the line marked 3 at the opposite end covers the region where the
nuclear lamina reside, adherent to the nuclear membrane). Nuclear
pores are indicated by arrows. Dilation of the endoplasmic reticulum (ER)
cisternae, a common effect of BFA, is also shown (4). B: WEHI-164 cells
1343
exposed to staurosporine (0.5 ␮M for 16 hr). Arrows indicate two apoptotic cells. Chromatin blocks can be visualized adhering to the nuclear
membrane (1). One of these blocks, in the cell on the left, has demilune
profile (2). C: Apoptotic HeLa cell exposed to BFA (2 ␮M) plus staurosporine (0.5 ␮M), both for 16 hr. Peripheral disposition of the chromatin
masses in the nucleus (1) and dilation of the ER cisternae (2) can be
observed.
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SESSO ET AL.
Fig. 3. A: An apoptotic cell from the secretory epithelium of the
prostate gland of a rat castrated 4 days previously is seen in the central
part of the micrograph. The nucleus is in the process of fragmentation
(1). The cytoplasm of the region at right is devoid of organelles (2), some
of which are well preserved and clustered at the opposite pole of the
profile (3). The arrow points to the region enlarged in the inset. B:
Apoptotic plasma cell from the granulation tissue of a scar experimentally induced 5 days previously in the dorsal skin of an adult rat. Besides
the two mitochondria exhibiting permeability transition (indicated by
arrows), a relatively large cluster of ⬃ 50 nm microvesicles can be
observed (1) (Sesso et al., 1999). C: Apoptotic macrophage from the
same granulation tissue referred to in B. A sector of the nucleus can be
observed in the upper right corner (1). The type 2 mitochondrion in the
lower central part (2) possesses a relatively small breach of the outer
mitochondrial membrane, indicated by an arrow.
ited incipient swelling. From 1% to 17% of the CUNS in
the various samples possessed mitochondria undergoing
the permeability transition. In these cases, many type 2 or
3 mitochondrial profiles could be found.
The successive morphological changes exhibited by
the apoptotic cells in the cultures we have examined
suggest that apoptotic cells undergo caspase-orchestrated lysis of all membrane-bound and cytoskeletal
MITOCHONDRIAL PERMEABILITY TRANSITION
1345
Fig. 4. A: Nonapoptotic pancreatic acinar cell from a pancreas that
had undergone ligature of the excretory ducts 2 days earlier. This cell
was close to others that were undergoing generalized cell death. Note
the normal texture of the chromatin (1) and the type 2 mitochondria
indicated by an arrow. B: Apoptotic pancreatic acinar cell from the same
gland referred to in A. Type 2 mitochondrion (indicated by the arrow) can
be visualized and, near the nucleus with compacted chromatin (1), a
cluster of ⬃ 50 nm microvesicles (2) is noted.
cytoplasmic structures. We observed dense chromatin
masses, often with circular profiles, in apoptotic nuclei
of cells in advanced stages of cytolysis. The cytoplasm of
these cells was either almost completely devoid of organelles or contained some dense or swollen type 2
mitochondria as residual organelles. When caspasedriven proteolysis and the action of nucleases was ad-
vanced, it was often difficult to be precise about the
former morphology of the nucleus and cytoplasmic organelles, all then appearing as remnants. These
changes occurred along with the complete rupture of the
cell membrane. When this happened, either in an apoptotic cell or in very rare, perhaps necrotic, nonapoptotic ones, the cytoplasm was fragmented and the or-
1346
SESSO ET AL.
Fig. 5. A and B are from PC-12* CUNS cells treated with BFA (2 ␮M)
and STS (0.5 ␮M) for 16 hr. The types of mitochondrial profiles are indicated
by numbers. In the upper left corner of A, a type 2 profile is sectioned at
various levels orthogonally to the plane of the image. Planes AB and CD
demonstrate type 2 and 3 profiles, respectively; plane EF, which contains
no cristae remnants, presents a type 4 profile. The arrows indicate unimembranous vesicles with homogeneous contents that do not resemble the
contents of the swollen part of profiles 2 that are devoid of cristae. These do
no look like type 4 profiles. In A, an asterisk marks a type 3 profile with a
relatively small breach of the outer membrane.
ganelles exposed to the culture medium became swollen
and disintegrated.
Two (PC-12* and WEHI-164 cells) of the four cell lines
(the two others are HL-60 and HeLa cells) in which the
mitochondria were studied morphometrically were also
subjected to various apoptogenic agents. In all eight samples, type 2 mitochondria were found in apoptotic cells
(Fig. 5) and in some of the cells with normal nuclei. Frequently, one observed type 2 mitochondrial profiles with a
relatively small site of rupture at the outer membrane
(lower type 2 profile in Fig. 3C and profile with an asterisk
in Fig. 5A). In the apoptotic cells, type 2 and 3 mitochondrial profiles may be present in the cytoplasm even in
advanced stages of cellular proteolysis. These cell profiles
exhibited a few scattered chromatin blocks and scarce
type 2 and 3 mitochondrial profiles in a cytoplasm virtually devoid of organelles. The main organelles in PC-12*
cells in advanced stages of apoptosis when the cell was
being segmented into apoptotic bodies were mitochondrial
profile types 2 and 3.
The configuration of the type 2 mitochondrial profiles
may vary considerable with regard to the degree of
swelling of the mitochondrial matrix. In Figure 6A and
6C, distinct differences in magnification are presented
to demonstrate that, in the smaller images (Fig. 6B and
6D), it is not easy to perceive whether the regions indicated by arrows actually have one or two membranes. In
these markedly swollen mitochondrial profiles (arrows
in Fig. 6B and D), two apposite poles are visible. One
appears as a dense membrane with fragments of cristae
nearby. Opposite to this pole, the mitochondrial matrix
is limited by a thin covering membrane. The region of
the profile with mitochondrial cristae and a dense membrane is where the profile exhibits both mitochondrial
membranes. At the opposite pole, the matrix is covered
only by the inner membrane.
MITOCHONDRIAL PERMEABILITY TRANSITION
1347
Fig. 6. Apoptotic HeLa and WEHI-164 cells, both exposed to STS
(0.5 ␮M) for 16 hr. These images illustrate that at relatively low magnifications, it is not possible to perceive that the dense parts of the
mitochondrial profiles close to the cristae and pointed by arrows in B
and D actually possess two mitochondrial membranes as shown in A
and C. The opposite pole of each enlarged profile is covered by a thin
inner mitochondrial membrane.
Surface-to-Volume Ratio of Type 1 and 2
Mitochondria in Apoptotic Cells and in
Companion Cells With Normal Nuclei
Evaluation of the volume, in ␮m3, and of the volume
fraction Vv, occupied by mitochondria types 1 and 2 in the
cytoplasm of apoptotic cells and in the corresponding
CUNS of the same cell culture, is presented in Table 1.
The data in the second line of Table 1 represent the best
estimates of the average volume per cell of types 1 and 2
mitochondria in PC-12* apoptotic cells and CUNS. The
mitochondria of the CUNS occupied 10% of the total cytoplasmic area or volume (second line/second column) and
had an average global volume per cell of 28 ␮m3. In apoptotic cells, mitochondrial profile type 1 and the sum of
types 2 and 3 occupied 2.3% and 3.8% (second line/columns 3 and 6, respectively) of the cytoplasm, with volumes of 6 and 10 ␮m3, respectively. In the PC12* apopto-
Measurements of the total surface area of the mitochondria carried out in the eight studied samples allowed us to
obtain the mitochondrial surface-to-volume ratio in all
apoptotic cells and CUNS examined (data not shown). The
ratio between the mitochondria of apoptotic cells and in
CUNS did not vary significantly (P ⬎ 0.05), indicating
that no important change in the average mitochondrial
volume between the two groups was actually detected.
Therefore, in these two groups, the mitocondrial volumes
are probably also a measure of the number of mitochondria per cell.
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SESSO ET AL.
tic cells, the volume of type 3 mitochondria is of 6 ␮m3,
representing 37.5% of the total mitochondrial volume of
these apoptotic cells (6/16 ⫻ 100 ⫽ 37.5%); in this case, the
value 16 ␮m3 (second line/column 7). The values of type 1
mitochondrial profiles found in apoptotic cells (column 3)
lied in the range of 14% (fifth line 3/22 ⫻ 100) to 53%
(sixth line 60/113 ⫻ 100) of the total mitochondrial volume
from these cells. In the various cell lineages analyzed, the
volumes associated with type 2 mitochondrial profiles varied from 25% to 77% of the estimated total mitochondrial
volume in apoptotic cells (column 4). For the apoptotic
PC-12* cells (second line/column 4), this value is 4/16 ⫻
100 ⫽ 25%. The percentages of the mitochondrial volume
of the apoptotic cells occupied by the sum of mitochondrial
profiles types 2 and 3 are presented in column 6. In the
apoptotic PC-12* cells of the second line, this percentage is
10/16 ⫻ 100 ⫽ 63%. In the various samples, this parameter varied from 47% (sixth line) to 89% (fourth line), with
an average of 69%. This important information conveyed
by the data of column 6 reveals that the majority of the
mitochondria of the apoptotic cells when captured under
the TEM are or were in the state of permeability transition. It was common to observe some 1–5 type 2 and some
2– 6 type 3 mitochondrial profiles in different sections of
apoptotic PC-12* cells.
The total mitochondrial volume of the apoptotic cells (v
in column 7) in six out of eight cases is lower than that of
the CUNS (lines 1, 2, 5, 6, 7, and 8 and columns 2 and 7 of
Table 1), while in the other two samples the mitochondrial
volume in apoptotic cells and in CUNS are about equal. In
the third line, the cited volume in apoptotic cells is 66
␮m3, while in the corresponding CUNS (column 2), it is 47
␮m3. If the 19 ␮m3 (see footnote to Table 1) of the CUNS
with type 2 mitochondria is included, the total amount is
equal to that of the apoptotic cells. In the fourth line, both
values of v (columns 2 and 7) for the mitochondrial volume
are equal (38 ␮m3).
As already mentioned, the incidence of type 2 mitochondria in cells with normal nuclei was quite variable (it
varied from 1% to 17%) and they would not always be
adequately sampled when 10 random (range, 10 –20) sections of cells with normal nuclei are taken for measurements. For this reason, with one exemption, presented as
an example (line 3 and footnote), we have not included
such cells in the nonapoptotic group when estimating mitochondrial volumes. None of the mitochondrial volumes
of the various columns of Table 1 correlate significantly
with the corresponding AIs. These varied from 6% to 68%.
Cytoplasmic Sectors Devoid of Organelles in
Apoptotic Cells and in CUNS
In many of the CUNS possessing a ruptured outer mitochondrial membrane, the cytoplasmic distribution of organelles was altered. The membrane-bound structures
were concentrated in certain sectors of the cytoplasm
while absent from others. This clustering of organelles is
more frequently found in apoptotic cells than in CUNS
(Fig. 3A). This observation suggests that the cytoskeleton
is undergoing alterations before the nuclear changes appear (data not shown). A literature search reveals that in
cells induced to undergo programmed death, alterations of
the distribution and morphology of the actin microfilaments is an early apoptotic event.
DISCUSSION
We decided to use transmission electron microscopy to
identify apoptotic cells as it seems to be the most reliable
procedure to accomplish this task (Yasuhara et al., 2003).
Morphological data to distinguish between necrotic and
apoptotic cells have long since been identified (Kerr et al.,
1995). A literature search revealed that the TUNEL reaction gives high false positive rates while DNA ladder assay lacks sensitivity (Yasuhara et al., 2003). The binding
of annexin V at the cell surface and positive staining with
propidium iodide occur in both apoptotic and necrotic
cells. In cultured cells, annexin V marks early stages of
apoptosis when the cell is still covered by a continuous
membrane that keeps the propidium iodide out of the cell.
As apoptosis progresses, various successive portions of
this membrane are removed, allowing the entrance of
propidium iodide that stains the cell. This apoptotic cell is
positive for both annexin V and propidium iodide, as are
necrotic cells (Vermes et al., 1995).
The results presented in this article clearly demonstrate
the high incidence of morphometric change in the mitochondria of apoptotic cells. From our data, we calculate
that at least 47– 89% (average 69%) of the mitochondria
from these cells have a ruptured outer membrane. Once
the outer mitochondrial membrane has ruptured, the inner membrane, covering an expanding swollen matrix,
passes through the formed hole and spreads into the surrounding cytoplasm. Since the continuous expansion of
the mitochondrial matrix needs to be membrane-bound in
order not to rupture, the membrane from the cristae are
incorporated into the inner membrane.
It is difficult to evaluate the magnitude of the underestimations of the actual volume of type 2 mitochondria. We
would need to know what fraction of the measured type 1
profiles derive from type 2 mitochondria and how the
unimembranous vesicles derived from type 2 mitochondria, but devoid of cristae, the type 4 profiles are numerically related to type 3 profiles. It was also evident during
the collection of morphometric data that the frequency of
the four profile types varied according to the morphological changes of the forming and developing type 2 mitochondria. The change in size of the unimembranous-bound
part of type 2 mitochondria will affect the yield of type 3
and 4 profiles when the mitochondria are randomly sectioned.
It is curious that, besides the publications of Angermüller et al. (1998) and of Kwong et al. (1999) in apoptotic
hepatocytes and secretory epithelial cells from the prostate gland of castrated rats, respectively, no other reports
of rupture of the outer mitochondrial membrane in apoptotic cells have been published.
As mentioned, in six samples out of eight, the mitochondrial volume of the apoptotic cells was less than that of the
CUNS. Since we do not know the magnitude of underestimation of the volume of type 2 mitochondria, we cannot
be sure of a possible reduction of this volume in apoptotic
cells. It must be noted that these results may simply
represent the process of organelle disassembly in many of
the cells analyzed. It is unclear whether the numerical
reduction of mitochondria detected in cells with typical
apoptotic nuclei had begun before the changes in nuclear
structure.
One of the facts that must have determined the lack of
statistical difference between the surface-to-volume ratio
MITOCHONDRIAL PERMEABILITY TRANSITION
of the mitochondria of the apoptotic cells and of the CUNS
is that, in cultures subjected to apoptogenic agents, the
CUNS often exhibit type 1 mitochondria with a spherical
shape (examples in Figs. 10 –15; Figs. 36, 72, 73, and 75 of
the site http://www.sebepa.cjb.net/). Not rarely a variable
part of these profiles, mainly in cultures with high AIs,
may appear swollen. It is possible that these swollen mitochondria found in CUNS, namely, those exhibiting ruptured outer membrane, are revealing that such cells are in
line to start showing the nuclear signs of apoptosis.
It is as yet unclear what causes the rupture of the outer
mitochondrial membrane in CUNS. The involvement of
terminal caspases such as caspase 3 can be excluded due
to the absence of nuclear alterations; these alterations are
a consequence of the activation of this caspase. It is also
unclear whether caspase 3 may influence the appearance
of type 2 mitochondria once the nuclear alterations have
begun. One cannot rule out the possibility that in many of
the observed cases an initiatory caspase, such as caspase
8, activates the mitochondrial-dependent apoptotic pathway (Scaffidi et al., 1998, 1999) and is a causative agent of
the rupture of the outer mitochondrial membrane in
CUNS. In this hypothesis, the type 2 mitochondria in
CUNS would be actively involved in causing the explicit
structural changes of apoptosis.
We suggest that type 2 mitochondria seems to be an
indicator of the MPT since the morphology of these swollen mitochondria could only be attained if the inner mitochondrial membrane had lost its selective permeability.
MPT has also been described in necrotic cells. However,
we do not know whether rupture of the outer mitochondrial membrane also occurs in necrotic cultured cells. In
rare identifiable necrotic cells found in our studies, the
cytoplasm was fragmented and the organelles, mitochondria included, in the process of disintegration. An answer
to this question may be obtained by performing TEM
studies in cells induced to undergo necrosis by an abrupt
and severe reduction in the cellular ATP levels (Kim et al.,
2003).
To associate the TEM images of type 2 mitochondria
undergoing MPT with the concept of a permeability transition pore or megachannel, it is necessary to envisage
that once the permeability transition pore is fully open, a
massive influx of fluids would occur between both mitochondrial membranes in the region of the pore, i.e., in a
restricted sector of the outer membrane. We have microscopic data that will be presented in a follow-up study,
supporting this assertion. As the punctual accumulation
of liquid progresses, it would promote a small focal rupture of the outer membrane. The depolarization of the
inner membrane would then rapidly spread from the initial point where the pore was. In such a manner, the
mitochondrial matrix would accumulate fluid from the
cytoplasm causing the swelling observed. The frequent
finding of type 2 mitochondrial profiles with relatively
small breaches in the outer membrane (see also results on
our Web site) supports this conjecture.
A clear-cut demonstration of rupture of outer mitochondrial membrane was obtained in mitochondria isolated
from cortical neurons and induced to undergo permeability transition. The release of cytochrome c from the intermembranous space appeared to be dependent of the rupture of the outer mitochondrial membrane (Brustovetsky
et al., 2002). A second occurrence of ruptured outer mitochondrial membrane in mitochondrial fractions induced to
1349
undergo permeability transition may be observed with a
magnifying lens in the right superior quadrant of Figure
4B from Petronilli et al. (1993) directly on the journal
page. Three extremely swollen and unequivocal type 2
mitochondrial profiles, joined two by two, may be seen.
The connection between the mitochondrial configuration
in apoptotic cells, and the results in isolated mitochondria
expressing permeability transition, substantiates the
view that the structurally altered mitochondria we are
observing in apoptotic cells and also in companion cultured cells with normal nuclei are actually expressing the
state of permeability transition.
Possibly in connection with the just cited observations
in isolated mitochondria, it is of relevance to mention that
the low-power images of type 2 mitochondrial profiles
shown in Figure 6B and 6D resemble transmission electron micrographs, similarly imaged, of pelleted swollen
mitochondrial induced to undergo MPT [Figs. 8B–D and
9A, C, and D in Beatrice et al. (1982); Fig. 4B and C in
Igbavboa and Pfeiffer (1988); Fig. 4B in Petronilli et al.
(1993); Fig. 3B in Jung et al. (1997)]. Ours and these
mitochondrial profiles have in common a dense region in
one pole of variable curved length. This dense region is
rapidly recognized by the presence of mitochondrial cristae close to or contacting its inner surface. In association
with a variable amount of cristae, the swollen isolated
mitochondria exhibit a more or less empty matrix with a
variable amount of cristae. These profiles opposite to their
dense region are covered by a thin membrane.
In order to clarify whether the type 2 mitochondria we
observe in CUNS and apoptotic cells actually release cytochrome c into the cytoplasm, we will carry out essays in
which the time-course incidence of type 2 mitochondria
will be correlated to the amount of cytosolic cytochrome c
in the cells under analysis. Parallel evaluation of the
activity of caspases 8, 9, and 3 will also be performed.
The following observations strongly suggest that the
rupture of the outer mitochondrial membrane we describe
is the mechanism by which intermembrane mitochondrial
proteins are released into the cytoplasm.
One, the morphology of the type 2 mitochondrial profiles
reveals exposure of the external surface of the inner membrane and therefore the intermembrane and membranous
mitochondrial proteins associated with this surface to the
cytoplasm. This indicates that there may be consequences
to the apoptotic program if cytochrome c is exposed to
cytoplasm containing the Apaf 1. If cytochrome c is indeed
loosely attached to the inner membrane (Lemasters et al.,
1998), it will be not only exposed, but actually progressively released into the cytoplasm as the inner membrane
transverses the breached outer membrane.
Two, various agents can open the permeability transition pore and promote loss of the inner mitochondrial
membrane selective permeability. The appearance of the
MPT is accompanied by the release of intermembrane
mitochondrial proteins that trigger the activation of procaspases (Tafani et al., 2001; de Giorgi et al., 2002; Kokoszka et al., 2004).
Three, the simultaneous occurrence of MPT and altered
organelle distribution in the cytoplasm of the CUNS is
another circumstantial element, compatible with the view
that in these cells the apoptotic program was already
beginning to take place.
Four, of the drugs we have employed to induce apoptosis
(listed in Table 1), staurosporine (Tafani et al., 2001), BFA
1350
SESSO ET AL.
(Ito et al., 2001), TNF (Vikhanskaya et al., 2002a), and
camptothecin (Stefanis et al., 1999; Sanches-Alcazar et
al., 2000) also promote the cytoplasmic release of cytochrome c.
ACKNOWLEDGMENTS
The authors thank Angela Batista Gomes dos Santos,
Maria Cecı́lia dos Santos L. Marcondes and Marcelo Alves
Ferreira of the Department of Pathology of the Faculty of
Medicini of São Paulo, for their technical support and
Miguel da Silva Passos Júnior from the Department of
Surgery for doing the photographic work.
LITERATURE CITED
Aherne WA, Dunnill MS. 1982. Morphometry. In: Arnold E, editor.
London. p 155–157.
Angermüller S, Kunstle G, Tiegs G. 1998. Pre-apoptotic alterations in
hepatocytes of TNFalpha-treated galactosamine-sensitized mice.
J Histochem Cytochem 46:1175–1183.
Antonsson B, Montessuit S, Sanchez B, Martinou JC. 2001. Bax is
present as a high molecular weight oligomer/complex in the mitochondrial membrane of apoptotic cells. J Biol Chem 276:11615–
11623.
Arcon LC, Taga R, Stomopoulos CD, Sesso A. 1980. Estimativa do raio
de estruturas esféricas a partir de medidas dos raios de cı́rculos em
cortes histológicos pelos métodos de Günter Bach. Ciência Cultura
32:1641–1653.
Bach G. 1963. Über die Bestimmung von charakteristschen Grossen
einer Kugelverteilung aus der Schnittkreise. Zeitschrift Wissenschaft Mikroskopie 65:285–291.
Basañez G, Nechushtan A, Drozhinin O, Chanturiya A, Choe E, Tutt
S, Wood KA, Hsu Y, Zimmerberg J, Youle RJ. 1999. Bax, but not
Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc Natl Acad Sci USA
96:5492–5497.
Beatrice MC, Palmer JW, Pfeiffer DR. 1980. The relationship between
mitochondrial membrane permeability, membrane potential, and
the retention of Ca2⫹ by mitochondria. J Biol Chem 255:8663– 8671.
Beatrice MC, Stiers DL, Pfeiffer DR. 1982. Increased permeability of
mitochondria during Ca2⫹ release induced by t-butyl hydroperoxide
or oxalacetate: the effect of ruthenium red. J Biol Chem 257:7161–
7171.
Belzacq AS, Vieira HL, Kroemer G, Brenner C. 2002. The adenine
nucleotide translocator in apoptosis. Biochimie 84:167–176.
Bernardi P. 1999. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79:1127–1155.
Brustovetsky N, Brustovetsky T, Jemmerson R, Dubinsky JM. 2002.
Calcium-induced cytochrome c release from CNS mitochondria is
associated with the permeability transition and rupture of the outer
membrane. J Neurochem 80:207–218.
Castedo M, Ferri K, Roumier T, Metivier D, Zamzami N, Kroemer G.
2002. Quantitation of mitochondrial alterations associated with
apoptosis. J Immunol Methods 265:39 – 47.
De Giorgi F, Lartigue L, Bauer MK, Schubert A, Grimm S, Hanson
GT, Remington SJ, Youle RJ, Ichas F. 2002. The permeability
transition pore signals apoptosis by directing Bax translocation and
multimerization. FASEB J 16:607– 609.
Desagher S, Martinou JC. 2000. Mitochondria as the central control
point of apoptosis. Trends Cell Biol 10:369 –377.
Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR. 2000. The
coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2:156 –162.
Gukovskaya AS, Perkins P, Zaninovic V, Sandoval D, Rutherford R,
Fitzsimmons T, Pandol SJ, Poucell-Hatton S. 1996. Mechanisms of
cell death after pancreatic duct obstruction in the opossum and the
rat. Gastroenterology 110:875– 884.
Gundersen HJ, Jensen EB. 1987. The efficiency of systematic sampling in stereology and its prediction. J Microsc 147(Pt 3):229 –263.
Gundersen HJ, Bendtsen TF, Korbo L, Marcussen N, Moller A,
Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A.
1988. Some new, simple and efficient stereological methods and
their use in pathological research and diagnosis. Acta Pathol Microbiol Immunol Scand 96:379 –394.
Ho PL, Raw I. 1992. Cyclic AMP potentiates bFGF-induced neurite
outgrowth in PC12 cells. J Cell Physiol 150:647– 656.
Hunter DR, Haworth RA. 1979. The Ca2⫹-induced membrane transition in mitochondria: III, transitional Ca2⫹ release. Arch Biochem
Biophys 195:468 – 477.
Igbavboa U, Pfeiffer DR. 1988. EGTA inhibits reverse uniport-dependent Ca2⫹ release from uncoupled mitochondria: possible regulation
of the Ca2⫹ uniporter by a Ca2⫹ binding site on the cytoplasmic side
of the inner membrane. J Biol Chem 263:1405–1412.
Ito Y, Pandey P, Mishra N, Kumar S, Narula N, Kharbanda S, Saxena
S, Kufe D. 2001. Targeting of the c-Abl tyrosine kinase to mitochondria in endoplasmic reticulum stress-induced apoptosis. Mol Cell
Biol 21:6233– 6242.
Jung DW, Bradshaw PC, Pfeiffer DR. 1997. Properties of a cyclosporin-insensitive permeability transition pore in yeast mitochondria. J Biol Chem 272:21104 –21112.
Kerr JF, Gobe GC, Winterford CM, Harmon BV. 1995. Anatomical
methods in cell death. Methods Cell Biol 46:1–27.
Kim JS, He L, Lemasters JJ. 2003. Mitochondrial permeability
transition: a common pathway to necrosis and apoptosis. Biochem
Biophys Res Commun 304:463– 470.
Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP,
MacGregor GR, Wallace DC. 2004. The ADP/ATP translocator is
not essential for the mitochondrial permeability transition pore.
Nature 427:461– 465.
Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M,
Schneiter R, Green DR, Newmeyer DD. 2002. Bid, Bax, and lipids
cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111:331–342.
Kuwana T, Newmeyer DD. 2003. Bcl-2-family proteins and the role of
mitochondria in apoptosis. Curr Opin Cell Biol 15:691– 699.
Kwong J, Choi HL, Huang Y, Chan FL. 1999. Ultrastructural and
biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration. Cell Tissue Res
298:123–136.
Kyprianou N, Isaacs JT. 1988. Activation of programmed cell death in
the rat ventral prostate after castration. Endocrinology 122:552–
562.
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura
Y, Crowe RA, Cascio WE, Bradham CA, Brenner DA, Herman B.
1998. The mitochondrial permeability transition in cell death: a
common mechanism in necrosis, apoptosis and autophagy. Biochim
Biophys Acta 1366:177–196.
Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ. 1995.
Contribution of the mitochondrial permeability transition to lethal
injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 307(Pt 1):99 –106.
Nieminen AL, Byrne AM, Herman B, Lemasters JJ. 1997. Mitochondrial permeability transition in hepatocytes induced by t-BuOOH:
NAD(P)H and reactive oxygen species. Am J Physiol 272:C1286 –
C1294.
Petit PX, Goubern M, Diolez P, Susin SA, Zamzami N, Kroemer G.
1998. Disruption of the outer mitochondrial membrane as a result
of large amplitude swelling: the impact of irreversible permeability
transition. FEBS Lett 426:111–116.
Petronilli V, Cola C, Massari S, Colonna R, Bernardi P. 1993. Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria. J Biol Chem
268:21939 –21945.
Pfeiffer DR, Schmid PC, Beatrice MC, Schmid HH. 1979. Intramitochondrial phospholipase activity and the effects of Ca2⫹ plus Nethylmaleimide on mitochondrial function. J Biol Chem 254:11485–
11494.
Ravagnan L, Roumier T, Kroemer G. 2002. Mitochondria, the killer
organelles and their weapons. J Cell Physiol 192:131–137.
Sanchez-Alcazar JA, Ault JG, Khodjakov A, Schneider E. 2000. Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat
cells. Cell Death Differ 7:1090 –1100.
MITOCHONDRIAL PERMEABILITY TRANSITION
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ,
Debatin KM, Krammer PH, Peter ME. 1998. Two CD95 (APO-1/
Fas) signaling pathways. EMBO J 17:1675–1687.
Scaffidi C, Schmitz I, Zha J, Korsmeyer SJ, Krammer PH, Peter ME.
1999. Differential modulation of apoptosis sensitivity in CD95 type
I and type II cells. J Biol Chem 274:22532–22538.
Sesso A, Fujiwara DT, Jaeger M, Jaeger R, Li TC, Monteiro MM,
Correa H, Ferreira MA, Schumacher RI, Belisario J, Kachar B,
Chen EJ. 1999. Structural elements common to mitosis and apoptosis. Tissue Cell 31:357–371.
Stefanis L, Park DS, Friedman WJ, Greene LA. 1999. Caspase-dependent and -independent death of camptothecin-treated embryonic
cortical neurons. J Neurosci 19:6235– 6247.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette
N, Prevost MC, Alzari PM, Kroemer G. 1999a. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J Exp Med
189:381–394.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N,
Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, Kroemer
G. 1999b. Molecular characterization of mitochondrial apoptosisinducing factor. Nature 397:441– 446.
Tafani M, Minchenko DA, Serroni A, Farber JL. 2001. Induction of the
mitochondrial permeability transition mediates the killing of HeLa
cells by staurosporine. Cancer Res 61:2459 –2466.
1351
Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT,
Thompson CB. 1997. Bcl-xL regulates the membrane potential and
volume homeostasis of mitochondria. Cell 91:627– 637.
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. 1995. A
novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled annexin V. J Immunol Methods 184:39 –51.
Vikhanskaya F, Falugi C, Valente P, Russo P. 2002. Human papillomavirus type 16 E6-enhanced susceptibility to apoptosis induced by
TNF in A2780 human ovarian cancer cell line. Int J Cancer 97:732–
739.
Walker NI, Bennett RE, Kerr JF. 1989. Cell death by apoptosis during
involution of the lactating breast in mice and rats. Am J Anat
185:19 –32.
Walker NI, Winterford CM, Williamson RM, Kerr JF. 1993. Ethionine-induced atrophy of rat pancreas involves apoptosis of acinar
cells. Pancreas 8:443– 449.
Yasuhara S, Zhu Y, Matsui T, Tipirneni N, Yasuhara Y, Kaneki M,
Rosenzweig A, Martyn JA. 2003. Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.
J Histochem Cytochem 51:873– 885.
Zamzami N, Kroemer G. 2003. Apoptosis: mitochondrial membrane
permeabilization—the (w)hole story? Curr Biol 13:R71–R73.
Zoratti M, Szabo I. 1995. The mitochondrial permeability transition.
Biochim Biophys Acta 1241:139 –176.
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