1 Center for Biosystems Research, University of Maryland Biotechnology
Institute, College Park, Maryland 20742, USA
2 Department of Cell Biology and Molecular Genetics, University of Maryland,
College Park, Maryland 20742, USA
* Author for correspondence (e-mail: baehreck{at}umbi.umd.edu)
Accepted 20 October 2003
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SUMMARY |
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Key words: Autophagic cell death, Apoptosis, Caspases, Salivary glands, Drosophila
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Introduction |
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Steroids are important regulators of programmed cell death in animals
(Baehrecke, 2000;
Distelhorst, 2002
;
Evans-Storm and Cidlowski,
1995
). During Drosophila development, the steroid
20-hydroxyecdysone (ecdysone) triggers cell death of many larval tissues,
including the larval salivary glands
(Jiang et al., 1997
).
Elevation of the ecdysone titer 12 hours after puparium formation (apf)
triggers autophagic cell death in the salivary gland, and this tissue is
completely destroyed by 16 hours apf
(Jiang et al., 1997
). Ecdysone
is bound by its heterodimeric receptor that is encoded by EcR and
usp, and acts in combination with the competence factor
ßFTZ-F1 (ftz-f1 FlyBase) to regulate
transcription of the primary response genes E93 (Eip93F
FlyBase), BR-C (br FlyBase) and E74
(Eip74EF FlyBase) (Broadus
et al., 1999
; Woodard et al.,
1994
). Salivary glands fail to die in animals with mutations in
ßFTZ-F1, E93, BR-C and E74A, and exhibit altered
transcription of the secondary response cell death genes rpr, hid
(W FlyBase), ark, dronc (Nc
FlyBase) and drice (Ice Flybase) during autophagic
cell death (Broadus et al.,
1999
; Jiang et al.,
2000
; Lee et al.,
2003
; Lee et al.,
2002b
; Lee et al.,
2000
; Restifo and White,
1992
).
The Drosophila genome contains cell death genes that have been
conserved between nematodes and humans
(Aravind et al., 2001;
Vernooy et al., 2000
).
Although the Drosophila cell death genes reaper
(rpr), head involution defective (hid),
grim and sickle appear novel
(Chen et al., 1996
;
Christich et al., 2002
;
Grether et al., 1995
;
Srinivasula et al., 2002
;
White et al., 1994
;
Wing et al., 2002
), the
proteins they encode contain limited but significant N-terminal sequence
identity with the mammalian proteins Omi/Htra2 and Smac/Diablo
(Wu et al., 2000
;
Wu et al., 2001
). These
conserved N-terminal sequences interact with the Drosophila inhibitor
of apoptosis DIAP1 (Hay et al.,
1995
), and this interaction is similar to the interaction of
Omi/Htra2 and Smac/Diablo with mammalian IAPs
(Wu et al., 2000
;
Wu et al., 2001
). The
interaction of Rpr, Hid, Grim and Sickle with DIAP1 relieves inhibition of
caspase proteases that then cleave substrates
(Goyal et al., 2000
;
Wang et al., 1999
). Seven
caspases have been identified in Drosophila, including Dronc, Dredd,
Dream/Strica, Decay, Daydream/Damm, Drice and Dcp-1
(Chen et al., 1998
;
Dorstyn et al., 1999a
;
Dorstyn et al., 1999b
;
Doumanis et al., 2001
;
Fraser and Evan, 1997
;
Harvey et al., 2001
;
Song et al., 1997
). Dronc,
Dredd and Dream/Strica are initiator caspases that can be activated in the
presence of the homolog of mammalian Apaf1 Ark/Dark/dApaf1/Hac-1
(Kanuka et al., 1999
;
Rodriguez et al., 2002
;
Rodriguez et al., 1999
;
Zhou et al., 1999
). Once
active, the initiatior caspases activate the effector or executioner caspases,
including Decay, Daydream/Damm, Drice and Dcp-1, that in turn cleave cell
substrates during programmed cell death.
Dying salivary gland cells contain autophagic vacuoles, but also appear to
use apoptosis genes, including caspases. Expression of the caspase inhibitor
p35 blocks salivary gland cell death and DNA fragmentation
(Jiang et al., 1997).
Furthermore, the apoptosis genes rpr, hid, ark, dronc and
drice are induced just before autophagic cell death
(Dorstyn et al., 1999a
;
Jiang et al., 1997
;
Jiang et al., 2000
;
Lee et al., 2003
;
Lee et al., 2002b
;
Lee et al., 2000
). These data
suggest similarities between autophagic cell death and apoptosis, but little
is known about how salivary gland cells are degraded. Salivary glands appear
to have the lysosomal machinery that is required to degrade their own
organelles, yet it is not clear whether they require the assistance of
phagocytes. Although salivary gland cells also appear to use genes that are
considered part of the core apoptotic machinery, including caspases, the
morphology of these cells and cells undergoing apoptosis are distinct.
Although caspase function is required for DNA fragmentation in dying salivary
glands (Lee and Baehrecke,
2001
), it is not known if these proteases cleave similar
substrates in cells that die by apoptotic and autophagic cell death.
Here we show that salivary gland destruction exhibits some similarities to
apoptosis, including blebbing and fragmentation of cells. Changes in the
levels of proteins, including nuclear Lamins and -Tubulin, are
regulated by caspase cleavage of these substrates. By contrast, filamentous
Actin appears to reorganize in a caspase-independent manner prior to
degradation. Animals with mutations in ßFTZ-F1, E93, BR-C and
E74A possess altered levels of active caspase, and altered levels of
structural proteins during salivary gland autophagy. Expression of either the
caspase inhibitor p35 or a dominant-negative form of the initiator caspase
Dronc is sufficient to prevent changes in nuclear Lamins and
-Tubulin,
but not in filamentous Actin. Combined, these studies indicate that caspases
and cytoskeleton reorganization are needed to drive the process of salivary
gland autophagic cell death.
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Materials and methods |
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Antibody staining
Antibodies against -Spectrin
(Dubreuil et al., 1987
), Lamin
Dm0 ADL84 (Stuurman et al.,
1995
),
-Tubulin 3A5
(Piperno and Fuller, 1985
),
Croquemort (Franc et al.,
1996
) and active Drice (Yoo et
al., 2002
) were obtained from Drs D. Branton, P. A. Fisher, M. T.
Fuller, N. C. Franc and B. A. Hay, respectively. Antibodies against cleaved
Caspase-3 (Asp175) and cleaved Lamin (Asp230) were obtained from Cell
Signaling Technology (Beverly, MA). Rhodamine Phalloidin, TOTO-3 and secondary
antibodies were purchased from Molecular Probes (Eugene, OR).
Wild-type Canton S, and ßFTZ-F1
(ßFTZ-F117/ßFTZ-F119),
BR-C (rbp5/Y), E74A
(E74AP[neo]/Df(3L)st-81k19) and E93
(E931/Df(3R)93Fx2) mutant salivary glands were
dissected from animals staged relative to puparium formation at 25°C,
fixed in 4% paraformaldehyde/heptane for 20 minutes at room temperature,
blocked in phosphate buffered saline containing 1% BSA and 0.1% Triton-X
(PBSBT), and incubated with primary antibodies for 16 hours at 4°C. The
ßFTZ-F1, BR-C, E74A and E93 mutants that were used in
this study and their general defects in salivary gland cell death have been
previously described (Broadus et al.,
1999; Lee and Baehrecke,
2001
; Lee et al.,
2000
; Restifo and White,
1992
). Following incubation with primary antibodies, salivary
glands were washed for 2 hours in PBSBT, incubated with appropriate secondary
antibodies for two hours at room temperature, washed for another 2 hours in
PBSBT at room temperature, incubated in 0.5 µl TOTO-3 in 1 ml PBSBT for 10
minutes at room temperature, and washed in PBSBT for an additional hour at
room temperature. For rhodamine Phalloidin staining of filamentous Actin,
salivary glands were fixed in 16% paraformaldehyde, incubated in 1% Triton-X
in PBS, and incubated in 5 µl of rhodamine Phalloidin in 100 µl PBS for
20 minutes at room temperature, as previously described
(Frydman and Spradling, 2001
).
Salivary glands were mounted in Vectashield (Vector Laboratories) and examined
using a Zeiss Axiovert 100 M confocal microscope. The confocal microscope
settings and length of exposure were identical in all analyses.
Expression of p35 and dominant-negative Dronc in salivary glands
To express p35 in salivary glands, y, w;
UAS-p35/UAS-p35 males were crossed to y,w,
fkh-GAL4/y,w, fkh-GAL4 virgin females. Progeny of this cross
were aged to 24 hours after puparium formation (apf) and stained for
expression of nuclear Lamin Dm0, Croquemort, -Tubulin,
-Spectrin
and filamentous Actin, as described above. To examine the impact of expressing
dominant-negative Dronc in salivary glands, transgenic males containing either
dominant-negative UAS-Dronc C318A
(Meier et al., 2000
) or
UAS-Dronc C318G (Quinn et al.,
2000
) were crossed to virgin females of either the salivary gland
GAL4 promoter strain y,w, fkh-GAL4/y,w, fkh-GAL4 or the
heat-inducible promoter strain y,w, hs-GAL4/y,w, hs-GAL4.
Controls consisted of dominant-negative Dronc transgenic strains that were not
crossed to a GAL4 driver strain. As the dominant-negative Dronc C318A strain
had a higher level of persistent salivary glands in preliminary studies, all
data that are reported were derived from this strain. Progeny derived from the
fkh-GAL4 and UAS-Dronc C318A cross were aged to 24 hours
apf, and analyzed for salivary gland persistence. Progeny from derived from
the hs-GAL4 and UAS-Dronc C318A cross were heat-shocked for
30 minutes at 37°C at 11, 13 and 16 hours apf, and analyzed for salivary
gland persistence. Persistent salivary glands were stained for expression of
nuclear Lamin Dm0, Croquemort,
-Tubulin,
-Spectrin and
filamentous Actin as described above.
Protein extraction and western blot analysis
Wild-type Canton S salivary glands were dissected from animals staged 8,
10, 12 and 14 hours apf, homogenized in 0.1% glycerol, 2% SDS, 0.125% 1M Tris
(pH 6.8), 0.05% ß-mercaptoethanol, and 0.05% Bromo-phenol blue, and
boiled for 5 minutes at 100°C. Equal amounts of total protein extracts
were separated by electrophoresis on 12% acrylamide gels, and either
transferred to 0.45 µm Immobilon-P membranes (Millipore), or duplicate gels
of identical extracts were assessed for equal loading and integrity by
Coomassie blue staining (Bio-Rad). Membranes were blocked in 10% non-fat milk
in PBS with 1% Tween 20 for 1 hour at 37°C, incubated in primary antibody
for 16 hours at 4°C, washed in PBS containing 1% Tween 20 at room
temperature, incubated with the appropriate HRP-conjugated secondary antibody
for 1 hour at 37°C, washed and developed using ECL detection reagents 1
and 2 (Amersham) at 25°C, and exposed to film.
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Results |
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Discussion |
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The mechanisms of cell degradation and removal appear to provide the
clearest distinction between apoptosis and autophagic cell death. In salivary
glands, dynamic changes in vacuole structure immediately precede their demise,
and such changes have not been reported in apoptotic cells
(Kerr et al., 1972;
Schweichel and Merker, 1973
).
Within one hour of salivary gland DNA degradation, large vacuoles appear to
break into smaller vacuoles, and this occurs within 2 hours of complete tissue
destruction (Lee and Baehrecke,
2001
) (Fig. 1). As
these large vacuoles fragment, smaller but distinct vacuoles accumulate near
the plasma membrane, and autophagic vacuoles containing components of the
cytoplasm, including mitochondria, are formed. Salivary gland cells then begin
to fragment, and nuclei and components of the cytoplasm then disperse within
the haemocoel (Fig. 2).
Although we can not exclude a role for phagocytes in autophagic cell death,
salivary gland cells proceed to late stages of degradation without the
assistance of phagocytes. It has been suggested that phagocytes may play a
secondary role in the removal of cellular debris towards the end of autophagic
cell death (Schweichel and Merker,
1973
), and our results are consistent with this possible
conclusion. The presence of autophagic vacuoles in degrading salivary glands
further indicates that autophagic cells use their own lysosomes for
degradation, whereas apoptotic cells depend on phagocytes for the bulk
degradation of long-lived cellular proteins.
Maintenance of the Actin cytoskeleton may be necessary for autophagic cell death
Dying cells exhibit dynamic changes in cell shape
(Kerr et al., 1972;
Schweichel and Merker, 1973
).
The changes in cell organization that occur during salivary gland cell death
are likely to be controlled by the modification of structural protein
organization. Indeed, dynamic changes in the abundance and localization of
filamentous Actin,
-Tubulin,
-Spectrin and nuclear Lamins
precede the death of salivary glands (Fig.
3). At least two possible explanations exist for how such changes
in protein expression are regulated in dying cells. One possibility is that
proteases cleave structural proteins
(Cryns and Yuan, 1998
), and
that this results in the changes in cell shape. Alternatively, changes in cell
shape could be regulated by the assembly of cytoskeletal proteins, such as
filamentous Actin, through signaling that is mediated by small GTPases
(Coleman and Olson, 2002
).
Proteolysis and changes in the assembly of the cytoskeleton both appear to
be involved in the regulation of changes that occur during autophagic cell
death of salivary glands. Although caspases play an important role in the cell
death of salivary glands, several lines of evidence suggest that some changes
in the structure of the cytoskeleton may occur in a caspase-independent
manner. First, whereas changes in filamentous Actin localization occur in
synchrony with changes in proteins such as nuclear Lamins that are cleaved by
caspases (Fig. 3), changes in
Actin protein levels are delayed by 4 hours
(Fig. 5). Second, mutations in
steroid-signaling genes, such as ßFTZ-F1, that prevent
expression of active caspase-3 and cleavage of nuclear Lamins do not prevent
changes in filamentous Actin localization
(Fig. 6). Third, although
inhibition of caspases by expression of either p35 or a dominant-negative form
of Dronc is sufficient to prevent changes in nuclear Lamins and
-Tubulin, these inhibitors are not sufficient to block changes in
filamentous Actin (Fig. 8).
These data are further supported by the observation that numerous small
GTPases increase their expression immediately prior to salivary gland cell
death (Lee et al., 2003
).
Although previous studies have suggested that changes in the Actin
cytoskeleton are required for autophagic cell death
(Bursch et al., 2000
;
Jochova et al., 1997
), the
failure to distinguish between cytoskeleton proteolysis and rearrangement has
made it difficult to interpret the potential significance of maintenance of
the cytoskeleton during cell death.
Several possibilities exist to explain why the cytoskeleton is maintained
during cell death. The cytoskeleton could be used to restrict the subcellular
location and activity of pro-apoptotic regulators of the Bcl-2 family, and
activation of apoptosis (Puthalakath et
al., 1999; Puthalakath et al.,
2001
). This mechanism seems unlikely during salivary gland cell
death because the Actin cytoskeleton is maintained after caspase-dependent
cleavage of substrates, including nuclear Lamins (Figs
3,
4,
5,
8). Alternatively, the Actin
cytoskeleton could be maintained as a substrate to localize proteins,
membranes and vacuoles within the cell. Intracellular trafficking plays an
important role in autophagy, as membrane-bound cytoplasmic components
(autophagic vacuoles) are transported to the lysosome for degradation
(Baehrecke, 2003
;
Klionsky and Emr, 2000
;
Ohsumi, 2001
). As we observe
autophagic vacuoles at stages after caspase activation and cleavage of
substrates such as nuclear Lamins (Lee and
Baehrecke, 2001
) (Figs
3,
4,
5), it is possible that the
Actin cytoskeleton is maintained to enable transport of vacuoles to
lysosomes.
Caspases are required for autophagic cell death
Studies of salivary glands indicate that caspases play an important role in
their autophagic cell death. The caspase-encoding genes dronc and
drice show an increase in their transcription following the rise in
steroid that triggers salivary gland autophagic cell death
(Lee et al., 2003;
Lee et al., 2002a
;
Lee et al., 2000
). This
increase in caspase transcription corresponds to the increase in active
caspase protein levels and in the cleavage of substrates such as nuclear
Lamins in dying salivary glands (Figs
4,
5). Mutations in the
steroid-regulated ßFTZ-F1, E93 and BR-C genes, which
prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice
expression, and have altered
-Tubulin,
-Spectrin and nuclear
Lamin expression in salivary glands (Figs
6,
7). Although E74A
mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice
levels and degraded nuclear Lamins (Fig.
7). Although these data are consistent with the partially degraded
morphology of E74A mutant salivary glands
(Lee and Baehrecke, 2001
), it
remains unclear what factor(s) E74A may regulate that are required
for normal cell death. However, our data indicate that ßFTZ-F1,
E93 and BR-C play a crucial role in determining caspase levels
in dying salivary gland cells, and this is supported by the impact of these
genes on the transcription of dronc
(Lee et al., 2002a
).
Significantly, inhibition of caspases by expression of either p35 or
dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in
nuclear Lamins and
-Tubulin, and death of salivary glands
(Jiang et al., 1997
;
Lee and Baehrecke, 2001
)
(Fig. 8).
The morphologies of dying cells indicate that apoptosis and autophagy are
distinct. However, the difference between these cells becomes less apparent
when one considers characteristics that were previously considered to be
specific to apoptosis. Clearly, markers such as DNA fragmentation, expression
and function of caspases, and cleavage of caspase substrates can exist in
cells that possess the morphology of autophagic cell death. As expression of
dominant-negative Dronc is sufficient to block caspase-dependent changes in
salivary glands, our studies also indicate that the mechanism for caspase
activation during autophagic cell death is similar to apoptosis during
Drosophila development; the initiator caspase Dronc regulates the
activation of the executioner caspase Drice and cleavage of cell substrates
(Yu et al., 2002). It is
surprising how little is known about the activity of caspases in developing
animals, as these proteases have been a subject of substantial investigation.
Recent studies indicate that caspases do not only function during autophagic
and apoptotic cell death, but that they are also used to degrade proteins
during the differentiation of sperm in Drosophila
(Arama et al., 2003
). Studies
of salivary glands indicate that the distinction between apoptosis and
autophagic cell death may be more subtle than their morphology suggests, and
raise the question of what makes these cells look so different. Restriction of
caspase activity within compartments of the dying cell may provide one
possible explanation, but it is also possible that other mechanisms of
proteolysis occur during autophagic cell death. This possibility is supported
by the large increase in transcription of non-caspase proteases just before
cell death of salivary glands (Lee et al.,
2003
), and by the fact that, unlike apoptotic cells that require
phagocyte lysosomes, salivary gland cells appear to degrade themselves through
autophagy. Future studies should provide important insights into the
similarities and differences in the mechanisms that regulate apoptosis and
autophagic programmed cell death.
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ACKNOWLEDGMENTS |
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