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First published online 5 March 2008
doi: 10.1242/dev.016295
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1 Cell Biology and Metabolism Program, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD 20892,
USA.
2 Institute of Biochemistry, Biological Research Center, Temesvari krt. 62,
H-6726, Szeged, Hungary.
3 Cell Cycle and Development Lab, Peter MacCallum Cancer Center, 7 St Andrews
place, East Melbourne, 3002 Victoria, Australia.
4 Department of Molecular Genetics and Microbiology, Duke University Medical
Center, Durham, North Carolina 27710, USA.
Author for correspondence (e-mail:
mlilly{at}helix.nih.gov)
Accepted 21 February 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: APC/C, Cdh1, Cyclin E, Drosophila, endoreplication, endocycle, Fzr/Cdh1, Geminin
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Traas et al.,
1998; Zybina and Zybina,
1996). The decoupling of S phase from mitosis makes the endocycle
a useful model for defining the minimum cell cycle requirements to achieve a
G/S oscillation and the associated once-per-cell-cycle replication of the
genome.
Current data strongly suggest that the Drosophila endocycle is
driven by the oscillations of Cyclin E/Cdk2 (Cdc2c - FlyBase) activity
(reviewed by Edgar and Orr-Weaver,
2001; Lilly and Duronio,
2005). In Drosophila, Cyclin E/Cdk2 activity is required
for DNA replication during both endocycles and mitotic cycles
(Knoblich et al., 1994). While
Cyclin E levels oscillate during endocycles, the continuous overexpression of
Cyclin E results in a block of endoreplication
(Follette et al., 1998;
Weiss et al., 1998). These
data are consistent with the model that endocycles require a Gap phase, when
overall Cyclin E/Cdk2 activity is low, in order to relicense DNA replication
origins for the subsequent S phase. Thus, the well-documented inhibitory
effects of Cyclin/Cdks on pre-replication complex formation observed during
the mitotic cycle (reviewed by Bell and
Dutta, 2002) appear to be conserved during endocycles. An
important factor controlling the periodicity of Cyclin E/Cdk2 activity is the
regulated accumulation and destruction of Cyclin E protein, as well as the
oscillations of the Cdk inhibitor Dacapo (Dap)
(Doronkin et al., 2003;
Hong et al., 2007;
Moberg et al., 2001). A
theoretical model, outlining the possible feedback relationships that might
control the periodicity of Cyclin E/Cdk2 activity during Drosophila
endocycles has been proposed (Edgar and
Orr-Weaver, 2001). However, the precise mechanism driving
endocycle progression and the accompanying oscillation of Cyclin E remains
unclear.
The mitotic kinase Cdk1 (Cdc2 - FlyBase) drives entry into mitosis. In
Drosophila, entry into the endocycle is contingent on the
downregulation of Cdk1 activity (Edgar and
Orr-Weaver, 2001; Lilly and
Duronio, 2005). Mutations in either Cdk1 or the positive
regulatory subunit Cyclin A, force cells undergoing mitotic cycles to enter
the endocycle (Hayashi, 1996;
Sauer et al., 1995;
Weigmann et al., 1997). The
mitotic cyclins Cyclin A, Cyclin B and Cyclin B3 are degraded by the highly
conserved Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase
that targets proteins for destruction by the 26S proteasome
(Sigrist et al., 1995;
Dawson et al., 1995;
Schaeffer et al., 2004;
Sigrist and Lehner, 1997).
Fizzy-related (Fzr; Rap - FlyBase) is a Cdh1-like positive regulatory subunit
of the APC/C that promotes the degradation of the mitotic cyclins in G1
(Jacobs et al., 2002;
Sigrist and Lehner, 1997;
Schaeffer et al., 2004). In
fzr loss-of-function mutants, cells fail to enter the endocycle
(Sigrist and Lehner, 1997;
Schaeffer et al., 2004).
Additionally, when overexpressed, fzr downregulates the levels of
Cyclin A, Cyclin B and Cyclin B3, and inhibits entry into mitosis
(Schaeffer et al., 2004;
Sigrist and Lehner, 1997).
Consistent with a role in promoting entry into the endocycle, fzr is
transcriptionally upregulated in the salivary gland, as well as the follicle
cells of the ovary, at the time of the mitotic/endocycle switch
(Shcherbata et al., 2004;
Sigrist and Lehner, 1997).
Taken together, these data indicate that an important step towards entering an
endocycle is the APC/CFzr/Cdh1-dependent destruction of the mitotic
cyclins.
However, although the requirement for the APC/C to enter the endocycle is
well established, its role beyond the mitotic/endocycle transition remains
unclear (Bentley et al., 2002;
Edgar and Orr-Weaver, 2001;
Kashevsky et al., 2002).
Because of the requirement for APC/C activity to switch from the mitotic cycle
to the endocycle, it has been difficult to assess whether the APC/C
contributes to the cell cycle oscillator that drives endoreplication. Current
models on the function of the APC/C during the endocycle are based on the
analysis of hypomorphic alleles of APC/C subunits that do not allow the
separation of these two potentially temporally independent functions
(Bentley et al., 2002;
Kashevsky et al., 2002;
Schaeffer et al., 2004;
Shcherbata et al., 2004). One
of the few exceptions where the APC/C is known to function during the
endocycle is in the polyploid nurse cells of the ovary
(Kashevsky et al., 2002;
Reed and Orr-Weaver, 1997). In
endocycling nurse cells, a transient increase in mitotic activity in stage 5
egg chambers initiates a developmental alteration in chromatin structure and
nuclear organization (Dej and Spradling,
1999; Kashevsky et al.,
2002; Reed and Orr-Weaver,
1997). In mutants that compromise APC/C activity, this transient
increase becomes permanent and the nurse cells arrest in a metaphase-like
state with high levels of mitotic cyclins
(Reed and Orr-Weaver, 1997).
These data support a model in which the APC/C restrains nurse cells from fully
entering the mitotic cycle after the brief burst of mitotic activity in the
fifth endocycle (Kashevsky et al.,
2002; Reed and Orr-Weaver,
1997). However, because the effect of compromising the APC/C in
nurse cells is restricted to a specific developmental stage and cell type, it
has been difficult to assess whether these results reflect a general
requirement for the APC/C in endoreplication. Thus, the precise importance of
the APC/C in promoting cell cycle progression during the endocycle remains to
be determined.
Here, we demonstrate that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. We show, using several strategies, that compromising APC/CFzr/Cdh1 activity after cells enter the endocycle results in the accumulation APC/C targets and a block to endoreplication. Moreover, our data support a model in which oscillations of APC/CFzr activity during the endocycle are driven by the periodic expression of Cyclin E.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), morula1/SM6a and
morula2/SM6a were obtained from the Bloomington Stock
Center. The Act>Cd2,f+>Gal4, hs-flp;; UAS-lacZ/TM6
Tb (Hazelett et al.,
1998) and hs-flp;; UAS-GFP, Act>Cd2>Gal4/TM3 Sb
(Shcherbata et al., 2004) were
gifts from Jessica Treisman and Hannele Ruohola-Baker, respectively. Christian
Lehner kindly provided the following overexpression lines: UAS-CycE III,
UAS-Fzr II and UAS-Fzy II
(Sigrist and Lehner, 1997).
hs-Rca1 lines (Dong et al.,
1997) were provided by Barbara Thomas. The UAS-Geminin
line used was UAS-Geminin III
(Quinn et al., 2001).
Generation of transgenic flies
The RNAi constructs against geminin and the individual
APC/C subunits Apc6, Apc8 and Apc10 were cloned in the pWIZ
vector, (Lee and Carthew,
2003). The pWIZ vector is under the control of a
GAL4-sensitive UAS enhancer, thus allowing the conditional or tissue
specific silencing of target genes. The constructions of the
Apc6RNAi and Apc8RNAi lines have been
described previously (Pal et al.,
2007). To generate the UAS-Apc10RNAi
construct, a 443 bp region was amplified by PCR using sequences corresponding
to exon 2 of the Apc10 transcript. The PCR product was cloned in
opposite orientations on both sides of the white intron present in the vector
(Lee and Carthew, 2003). To
generate the UAS-gemininRNAi construct, a region of 500 bp
of geminin cDNA was amplified by PCR and then two copies of this sequence were
cloned in opposite orientations into the pWIZ vector. Two transgenic
lines were generated, M4-gemininRNAi and
M6-gemininRNAi, of which M4 was shown to result in a more
complete knockdown of Geminin protein (data not shown). In this study, we used
the M4-gemininRNAi line. To generate the
UASp-Rca1 construct, the 1.3 kb BstBI-AseI cDNA
fragment containing the entire rca1-coding region and poly(A) signal
provided by the 
-tubulin 3'-untranslated region (UTR) were
inserted into the pUASP(-) vector for P-element transformation. pUASP(-) is a
derivative of pUASP vector (Rorth,
1998) and lacks 1.3 kb K10 3'-UTR. Construction of
the transgene of Orc1-GFP fusion driven by 2.4 kb orc1
promoter has been described previously
(Araki et al., 2003). The
stable Orc1 derivative transgene was prepared by the alanine substitution of
L295 and N299 in the O-box that mediates Fzr-specific APC/C degradation
(Araki et al., 2005).
Generation of flip-out clones
Somatic overexpression was achieved by generating Flip-out/Gal4 clones
(Pignoni and Zipursky, 1997)
in hs-flp/+; Act>CD2>Gal4, UAS-GFP/+; UAS-x/+ larvae or females
(x denotes Apc6RNAi, Apc8RNAi,
Apc10RNAi, GemininRNAi, Geminin, CycE, fzr
or fzy. Clones were induced by heat-shocking larvae or females at
37°C for 15 minutes. Subsequently, early L3 stage larvae were dissected 3
days later and adult females were dissected 1 day later. Using this strategy,
we were able to induce clones in cells after they had entered the endocycle.
We verified that spontaneous clones are rare (3.1% of non heat-shocked
ovarioles have one follicular clone, n=221 ovarioles). Clones
expressing Gal4 were induced by `flipping out' an interruption cassette
Act>CD2>Gal4 transgene in a genetic background that contained the UAS-x
constructs, as well as a UAS-GFP or UAS-lacZ transgene. Thus, the
co-expressing GFP or β-galactosidase marks cells that express the UAS-x
constructs. For the hs-Rca1 experiment in the nurse cells, females were
heat-shocked at 37°C for 1 hour and dissected 1 day later.
Immunocytochemistry
Immunocytochemistry of adult ovary staining was performed as described
previously (McKearin and Ohlstein,
1995). The following antibodies were used in this study: mouse
monoclonal -GFP 1:200 (Roche), rabbit polyclonal -GFP 1:500
(Molecular Probes), mouse monoclonal -β-Galactosidase 1:500
(Promega), mouse monoclonal -Cyclin A A12 1:100
(Sigrist et al., 1995), mouse
monoclonal -Cyclin B F2F4 1:50
(Lehner and O'Farrell, 1990),
rat polyclonal -Geminin 1:1000
(Quinn et al., 2001) and
purified rat polyclonal -Orc1 1:30
(Asano and Wharton, 1999).
Rabbit polyclonal -Cyclin A antibody 1:50
(Lehner and O'Farrell, 1989)
was a gift from Christian Lehner. -Geminin staining was performed using
two independent antibodies (Calvi et al.,
1998; Quinn et al.,
2001). Both -Geminin antibodies gave similar staining
patterns in both the embryo and the ovary (data not shown).
Fluorescence-conjugated secondary antibodies were purchased from Molecular
Probes and were used at a 1:1000 dilution. All samples were mounted in
cytifluor (Kent). Samples were examined with an Olympus Fluoview FV1000
microscope and composite figures were prepared using Adobe Photoshop 7.0.
Semi-quantitative RT-PCR
The semi-quantitative RT-PCR was carried out according to
Le and Richardson, 2004
(Le and Richardson, 2004).
Salivary glands from hs-flp/+; Act>CD2>Gal4, UAS-GFP/+;
UAS-CycE/+ and hs-flp/+; Act>CD2>Gal4,
UAS-GFP/UAS-Apc10RNAi; UAS-Apc8RNAi/+ larvae were
heat-shocked at 37°C for 1 hour twice to ensure that a large number of
cells overexpress the CycE or ApcRNAi constructs.
Total RNA was extracted from 4- to 8-hour embryos and third instar larval
salivary glands using Trizol reagent (Invitrogen) according to the
manufacturer's protocol. Reverse transcription-polymerase chain reaction
(RT-PCR) was performed using the SuperScript II Reverse Transcriptase
(Invitrogen) according to the manufacturer's protocol to produce the cDNA
(first strand). Each cDNA was primed by random hexamer (Gibco). The PCR was
performed according to standard methods by using a PTC-200 Peltier thermal
cycler. RT-PCR amplicons were in the linear phase of amplification. Expression
of the ribosomal protein 49 (rp49; RpL32 - FlyBase)
gene was used for normalization. The bands were quantified using Quantity One
software (BioRad).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Gonzalez et al., 2006). In
Drosophila, Geminin was reported to be present in some endocycling
cell types (Quinn et al.,
2001). However, upon re-examination we found that, as is observed
in mammals, Geminin levels are dramatically downregulated upon entry into the
endocycle. In the follicle cells of the ovary, Geminin levels are reduced,
coincident with entry into the first endocycle in stage 6 of oogenesis
(Fig. 1A,A', arrow).
Additionally, Geminin levels are low as the nurse cells enter the endocycle in
region 3 of the germarium (Fig.
1A,A', inset, arrowhead). Consistent with what is observed
in the ovary, during embryogenesis Geminin levels are dramatically
downregulated as cells enter the endocycle in multiple cell types. Geminin is
present at high levels early in embryogenesis during the syncytial divisions
and cellularization, irrespective of cell cycle stage
(Quinn et al., 2001) (see Fig.
S1A,A' in the supplementary material). Thereafter, Geminin is expressed
in mitotically dividing cells, including the central nervous system cells
(CNS) and brain lobe cells (BL) (Fig.
1B,B') (Quinn et al.,
2001). By contrast, Geminin levels are low in the population of
cells that have entered the endocycle, including the embryonic salivary gland
(SG), the midgut (MG), the hindgut (HG) and the Malpighian tubules (MT)
(Fig. 1B,B'). Geminin
remains low throughout the process of endoreplication, with low levels of the
protein observed in the endocycling cells of the larval salivary glands
(Fig. 1C,C') and
Malpighian tubules (see Fig. S1B,B' in the supplementary material).
Thus, as is observed in mammalian trophoblasts, in Drosophila the
levels of Geminin are downregulated at the mitotic/endocycle transition.
The downregulation of Geminin observed upon entry into the endocycle
mirrors the behavior of the APC/C targets Cyclin A and Cyclin B
(Lehner and O'Farrell, 1989;
Lilly and Spradling, 1996;
Reed and Orr-Weaver, 1997;
Sauer et al., 1995).
morula (mr) encodes the APC/C subunit APC2
(Kashevsky et al., 2002). In
mr/Apc2 hypomorphic female-sterile mutants, nurse cells proceed
through the first four rounds of endocycle in a manner indistinguishable from
wild type, but in stage 5 of oogenesis, the nurse cells condense and arrest in
a metaphase-like state (Reed and
Orr-Weaver, 1997). This arrest is accompanied by the accumulation
of the mitotic cyclins Cyclin A and Cyclin B
(Kashevsky et al., 2002;
Reed and Orr-Weaver, 1997;
Sugimura and Lilly, 2006). We
find that Geminin is also ectopically expressed in the polyploid nurse cells
in mr1/mr2 mutants
(Fig. 1D,D').
Intriguingly, the increase in Geminin levels in mr/Apc2 mutants is
observed much earlier in oogenesis, in region 3 of the germarium, than the
rise in the levels of the mitotic cyclins, which is not observed until stage 5
(Reed and Orr-Weaver, 1997)
(Fig. 1D,D', inserts,
arrow). Additionally, Geminin levels are increased in the endocycling cells of
the larval salivary gland in mr/Apc2 mutant larvae
(Fig. 1E,E'). Thus, as is
observed with the mitotic cyclins, the APC/C functions to keep Geminin levels
low during Drosophila endocycles.
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;
Reed and Orr-Weaver, 1997).
However, it was unclear whether this requirement was cell type specific, only
being necessary to progress through the unusual pseudomitotic-like fifth
endocycle that occurs in nurse cells
(Kashevsky et al., 2002) or
whether APC/C activity was generally required for endocycle progression. To
examine this issue, we used an inducible RNAi system to knockdown the levels
of several APC/C subunits after cells had successfully entered the endocycle.
This strategy, which uses the inducible hs-FLP/Gal4 (Flipout/Gal4) system
(Pignoni and Zipursky, 1997),
allows us to examine the role of the APC/C during endocycles, independent of
the requirement for APC/C activity to transit the mitotic/endocycle boundary.
To examine the role of the APC/C during endocycles in the salivary gland, RNAi
constructs are induced during the L1 larval stage, after cells have initiated
endocycles (Smith et al.,
1993) (Fig. 2A). In
our studies we used RNAi constructs against Apc6/Cdc16 and Apc8
(Pal et al., 2007) and Apc10.
Apc6 and Apc8 are tetratrico peptide repeat (TPR) subunits, which, in humans,
are part of the core APC/C complex
(Vodermaier et al., 2003). In
budding yeast, Apc10/Doc1 is required for the processivity of APC/C-mediated
ubiquitination reactions (Carroll and
Morgan, 2002) and for substrate binding
(Passmore et al., 2003).
When expressed in the salivary gland after cells have entered the
endocycle, all three ApcRNAi constructs result in the
accumulation of the APC/C targets Geminin, Cyclin A and Cyclin B (Fig.
2B-B''',
2C-C'''; see Fig. S2 and
Table S1 in the supplementary material). Additionally, although no abrogation
of the endocycle was associated with the expression of
Apc6RNAi or Apc10RNAi (Fig. S2 in the
supplementary material), we observe a notable decrease in the size of salivary
gland nuclei, as measured by DAPI staining, in a proportion of cells (20.6%,
n=218) that express the Apc8RNAi construct
(Fig. 2B'', arrowheads).
Moreover, the co-expression of the two ApcRNAi constructs,
Apc8RNAi and Apc10RNAi, results in a
dramatic decrease in nuclear size in a large fraction (42.9%, n=291)
of salivary gland nuclei from early third instar larvae
(Fig. 2D-D'', arrows).
Consistent with the reduced ploidy values observed with the expression of
Apc8RNAi, we find that the APC/C targets Cyclin A and
Geminin accumulate to higher levels after the expression of
Apc8RNAi relative to what is observed after the expression
of Apc6RNAi or Apc10RNAi (see Table S1
in the supplementary material). Thus, the minimal effect on DNA content
observed after Apc6RNAi and Apc10RNAi
expression correlates with reduced APC/C target accumulation. There are
several possible explanations for why Apc8RNAi shows a
more robust response. First, this may reflect the reduced on target efficiency
of the Apc6RNAi and Apc10RNAi
constructs relative to the Apc8RNAi. Alternatively, the
Apc6 and Apc10 proteins may have increased stability relative to the Apc8
protein when assembled in the large multiprotein APC/C complex
(Davis et al., 2002;
Shakes et al., 2003). These
data support the model that the APC/C functions beyond the mitotic/endocycle
transition to promote endoreplication.
In somatic tissues, the APC/C has two potential activators: Fizzy/Cdc20
(Fzy/Cdc20) and Fizzy-related/Cdh1 (Fzr/Cdh1)
(Dawson et al., 1995;
Sigrist et al., 1995;
Sigrist and Lehner, 1997). In
Drosophila, Fzr/Cdh1 but not Fzy/Cdc20, is required for cells to
enter the endocycle (Sigrist and Lehner,
1997). Previous works indicate that APC/CFzr/Cdh1
activity is downregulated by Cyclin E/Cdk2
(Sigrist and Lehner, 1997). We
find that in both the salivary gland and follicle cells of the ovary, the
overexpression of Cyclin E after cells have entered the endocycle results in
the accumulation of the APC/C targets Geminin
(Fig. 3A-A''',C-C''),
Cyclin A (Fig. 3B-B'') and
Cyclin B (data not shown). Consistent with the model that the accumulation of
these APC/C targets is due to the Cyclin E-dependent inhibition of Fzr/Cdh1,
the co-expression of Fzr/Cdh1 with Cyclin E prevents the accumulation of these
APC/C targets during follicle cell endocycles
(Fig. 3D-D''). By
contrast, the co-expression of Fzy/Cdc20 with Cyclin E has no effect on
preventing the Cyclin E-induced accumulation of the APC/C targets Cyclin A and
Geminin in follicle cells (data not shown). In the larval salivary gland, the
co-expression of Fzr/Cdh1 with Cyclin E did not inhibit the expression of
APC/C targets (data not shown). This may reflect the different time frame over
which endoreplication occurs in the follicle cells versus salivary glands.
Although most follicle cells undergo only three endocycles to attain ploidy
values of 16C, many of the cells in the salivary gland undergo at least 10
endocycles to achieve ploidy values of greater than 2000C
(Hammond and Laird, 1985a;
Hammond and Laird, 1985b;
Lilly and Spradling, 1996).
Cyclin E/Cdk2 acts catalytically to inhibit Fzr/Cdh1, whereas the ability of
Fzr/Cdh1 to activate the APC/C is achieved stoichiometrically
(Passmore and Barford, 2005).
Therefore, we reasoned that the ability of Fzr to overcome inhibition by
Cyclin E/Cdk2 activity might diminish as Cyclin E levels increase over time.
Thus, the accumulation of APC/C targets in UAS-Cyclin E; UAS-Fzr
salivary glands may reflect the inability of the exogenously supplied Fzr/Cdh1
to overcome the inhibitor effects of Cyclin E in this time frame.
|
;
Grosskortenhaus and Sprenger,
2002). In rca1 mutants, mitotic cells are rushed into an
endocycle, presumably because of unrestrained APC/CFzr/Cdh1
activity, which prevents the accumulation of the mitotic cyclins and
progression into mitosis (Dong et al.,
1997; Grosskortenhaus and
Sprenger, 2002). In both the salivary gland and the follicle cells
the expression of Rca1 after cells have entered the endocycle, using
the FLPout/Gal4 technique, results in the accumulation of APC/C targets
(Fig.
4A-A'',B-B'',C-C''). Additionally, using a
transgenic line that contains Rca1 under the control of the inducible
heat-shock promoter, we find that the induction of Rca1 expression
results in the accumulation of APC/C targets in the endocycling nurse cells
(Fig. 4D-D''). The
observation that expressing an inhibitor of the APC/CFzr/Cdh1
triggers the accumulation of APC/C targets in multiple polyploid cell types
further supports the model that APC/CFzr/Cdh1 functions beyond the
mitotic/endocycle transition.
Geminin is not the only essential downstream target of the APC/C during endocycles
In Drosophila, the overexpression of Cyclin E results in a block
to S phase during endocycles but not mitotic cycles
(Follette et al., 1998;
Weiss et al., 1998). As
described above, we find that the overexpression of Cyclin E in the
endocycling cells of the salivary gland, results in the accumulation of APC/C
targets, including the Cdt1/Dup inhibitor Geminin
(Fig. 3A-A''',C-C'').
Because of its ability to inhibit the activity of the licensing factor
Cdt1/Dup, the overexpression of Geminin blocks DNA replication
(Fig. 5A-A''')
(McGarry and Kirschner, 1998;
Quinn et al., 2001).
Therefore, we reasoned that the block to endoreplication observed upon the
overexpression of Cyclin E might be due to the unscheduled accumulation of
Geminin. In order to test this hypothesis, we co-expressed a
UAS-GemininRNAi construct with UAS-Cyclin E using
the Flipout/Gal4 system (hs-flp/+; UAS-GemininRNAi/+;
Act>CD2>Gal4, UAS-GFP/UAS-CycE larvae). Whereas the overexpression
of Cyclin E results in accumulation of Geminin in the cells of the salivary
gland, the co-expression of GemininRNAi with Cyclin E
reduced Geminin below the level of detection
(Fig. 5B'). However,
preventing the accumulation of Geminin did not rescue the block to DNA
replication associated with the constitutive expression of Cyclin E [compare
the wild-type nucleus (arrowhead) to the UAS-GemininRNAi;
UAS-CycE nucleus (arrow) in Fig.
5B'']. This may reflect the ability of high Cyclin E/Cdk2
activity to inhibit DNA replication origins directly during the endocycle
and/or the presence of additional important APC/C targets that must be
destroyed in order to facilitate endocycle progression.
|
|
).
APC/CFzr/Cdh1 drives the oscillation of Orc1 during the endocycle
Origin Recognition Complex 1 (Orc1) is a highly conserved component of the
pre-replication complex (pre-RC) (Dutta
and Bell, 1997). The levels of the Orc1 protein oscillate during
the nurse cell and follicle cell endocycles
(Asano and Wharton, 1999).
Consistent with these earlier observations, we find that Orc1 levels oscillate
during the endocycle in larval salivary glands
(Fig. 6F). Intriguingly, the
constitutive transcription of an Orc1 transgene from a heterologous promoter
does not notable diminish the kinetics of Orc1 protein oscillations during
endocycles (Araki et al.,
2005). Moreover, Orc1 oscillations during the endocycle do not
require the native Orc1 3' or 5' UTR, and therefore are unlikely
to be controlled at the level of translation
(Araki et al., 2005). In
Drosophila, Orc1 is a target of APC/CFzr/Cdh1
(Araki et al., 2003;
Araki et al., 2005). We find
that compromising APC/C activity in salivary gland cells using either the
ApcRNAi strategy as outlined in
Fig. 2A
(Fig. 6A-A'') or by
overexpressing Cyclin E (Fig.
6B-B'') results in the increased accumulation of Orc1
protein. Indeed, 65% of the cells that express the
Apc10RNAi; Apc8RNAi constructs have
high levels of Orc1 (n=63, Fig.
6A), compared with 14% of wild-type nuclei (n=261,
Fig. 6B, arrow).
|
-Orc1 and
-MPM2 antibodies. Although traditionally used to follow mitotic
phosphoepitopes, the -MPM2 antibody has proven a useful marker for
monitoring Cyclin E/Cdk2 activity in Drosophila, with the presence of
one or more -MPM2-positive subnuclear spheres, which have recently been
shown to be histone bodies (White et al.,
2007), correlating with high levels of Cyclin E/Cdk2 activity
(Calvi et al., 1998;
Royzman et al., 1999). We find
that high levels of Orc1 strongly correlate with high levels of -MPM2
histone body staining in the endocycling nurse cells
(Fig. 6E-E'' and
Table 1) and follicle cells
(see Fig. S3B-B'' in the supplementary material and
Table 1) of the ovary, as well
as in the larval salivary gland (Fig.
6F-F'' and Table
1). These data support the model that Cyclin E/Cdk2 activity, and
the subsequent inhibition of the APC/CFzr/Cdh1, may drive the
periodic accumulation of Orc1 during wild-type endocycles.
|
). The O-box
is a novel APC/C targeting motif that is necessary and sufficient for
directing the Fzr/Cdh1-dependent degradation of Orc1
(Araki et al., 2005). In
endocycling salivary glands, nurse cells and follicle cells,
porc1-Orc1-GFP levels oscillate in a manner similar to that observed
with native Orc1 protein (Fig.
6C, compared with
6F). By contrast,
porc1-Orc1Oboxmut-GFP protein levels are increased in salivary glands
(Fig. 6D), nurse cells (Fig.
S3A) and follicle cells (see Fig. S3C in the supplementary material).
Specifically, in the nurse cells the levels of porc1-Orc1Oboxmut-GFP
protein are uniformly high (see Fig. S3A in the supplementary material), while
in the follicle cells and salivary glands there is some cell-to-cell variation
in porc1-Orc1Oboxmut-GFP levels (see Fig. S3C in the supplementary
material; Fig. 6D). Does this
cell-to-cell variation indicate that cell cycle-dependent oscillations of Orc1
can occur independently of O-box-mediated destruction by APC/CFzr?
In order to answer this question, we double-labeled ovaries and salivary
glands with -GFP and -MPM2 antibodies. In the follicle cells,
nurse cells and salivary gland cells, we find that
porc1-Orc1Oboxmut-GFP levels do not correlate with -MPM2
histone body staining (Table
1), indicating that cell cycle regulation of Orc1 levels is O-box
dependent. Our data do not eliminate the possibility that there are additional
inputs that influence the stability of the Orc1 protein during endocycles.
However, taken together, our data strongly suggest that the oscillation of
Orc1 during the endocycle is at least partially driven by the O-box-mediated
destruction of the Orc1 protein by APC/CFzr/Cdh1.
|
;
Lehner and O'Farrell, 1990;
Whitfield et al., 1989). We
used semi-quantitative RT-PCR to compared the transcript levels for Cyclin
A, geminin and Orc1, in 4- to 8-hour-old embryos (primarily
cells in the mitotic cycle) with the levels of these transcripts in salivary
glands from early third instar larvae (primarily cells in the endocycle).
Consistent with previous reports, we find that the levels of Cyclin A
transcript are reduced almost sevenfold in endocycling versus mitotic cells,
relative to the levels of the ribosomal protein 49 (rp49),
while the levels of geminin transcript are reduced approximately
threefold (see Fig. S4 in the supplementary material). By contrast, the levels
of the Orc1 transcript are only slightly downregulated. Intriguingly,
when a large number of heat shocks are performed to increase the number of
cells that express either the ApcRNAi constructs or
CycE in endocycling cells of the larval salivary gland, we observe a
modest increase in the transcript levels of Cyclin A and
geminin (see Fig. S5 in the supplementary material). Thus, these data
suggest that, perhaps as is observed in mammals, the regulation of APC/C
activity may drive a positive-feedback circuit that controls both protein
stability and mRNA expression (Verschuren
et al., 2007). However, as discussed in detail below, differences
in transcript levels is just one of several possible explanations for the
differential expression of APC/C targets during endocycles. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Lilly and
Duronio, 2005). This is accomplished, at least in part, by
preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and
Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic
cycle, the mitotic cyclins are periodically targeted for regulated proteolysis
by the E3-Ubiquintin ligase the APC/C
(Dawson et al., 1995;
Sigrist et al., 1995;
Sigrist and Lehner, 1997). Yet
the transcriptional downregulation of several APC/C targets at the
mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25,
suggested that the proteolytic activity of the APC/C might not be necessary
during endocycles (Lehner and O'Farrell,
1989; Lehner and O'Farrell,
1990; Whitfield et al.,
1989). However, we find that compromising APC/C activity, after
cells have entered the endocycle, results in the accumulation of Geminin and
the mitotic cyclins, and in a block of DNA replication. Thus, the
transcriptional downregulation of APC/C targets observed at the
mitotic/endocycle transition is either downstream of APC/C activity and/or not
sufficient to maintain low levels of these proteins. Taken together, our data
suggest a model in which APC/C promotes the G/S oscillation of the endocycle
by preventing the unscheduled accumulation of Geminin and the mitotic
cyclins.
We find that during endocycles, APC/C activity prevents the inappropriate
accumulation of Geminin, an inhibitor of the DNA replication-licensing factor
Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently
inhibits DNA replication (Fig.
5) (Quinn et al.,
2001). These results strongly suggest that an essential function
of the APC/C during the endocycle is to prevent the unregulated accumulation
of Geminin. A similar role has been proposed for the APC/C during
endoreplicative cycles of mouse trophoblasts
(Gonzalez et al., 2006).
However, our data indicate that Geminin is not the only essential target of
the APC/C during endocycles. A candidate for a second important target of the
APC/C during endocycles is Cyclin A. Previous studies have shown that the
overexpression of Cyclin A in the salivary gland, between the first and second
endocycle, results in variable inhibitory effects on endoreplication
(Weiss et al., 1998). Although
the majority of salivary gland cells that overexpress Cyclin A appear to be
unaffected, a small percentage show a marked decrease in ploidy values. The
reason for this variability is not clear
(Weiss et al., 1998). However,
if the inhibitory influence of Cyclin A is mediated through binding and
activation of Cdk1, this effect may be greatly amplified in the presence of
high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1
(Kumagai and Dunphy, 1991;
Strausfeld et al., 1991).
Recent studies indicate that String/Cdc25, which contains both a consensus Ken
box and D-box, is a target of the APC/C
(Donzelli et al., 2002;
Tanaka-Matakatsu et al., 2007)
(Barbara Thomas, personal communication). Therefore, an essential function of
the APC/C during endocycles may involve restricting the activity of the
mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and
String/Cdc25. Finally, we note that the APC/C may have additional essential
targets during the endocycle, which have yet to be identified.
The periodic accumulation of the Orc1 protein during endocycles strongly
suggests that the activity of the APC/CFzr/Cdh1 may not be
continuous but cyclical. Previous work indicates that in Drosophila
Cyclin E/Cdk2 inhibits the activity of APC/CFzr/Cdh1
(Reber et al., 2006;
Sigrist and Lehner, 1997).
These data are consistent with the observation that phosphorylation of
Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the
APC/C in yeast, Xenopus and mammals
(Kramer et al., 2000;
Zachariae et al., 1998).
During the endocycle, the levels of Cyclin E oscillate
(Follette et al., 1998;
Weiss et al., 1998). Taken
together, these observations suggest a model in which APC/CFzr/Cdh1
is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of
Cyclin E resulting in the inhibition of APC/CFzr/Cdh1 activity and
low levels of Cyclin E permitting full APC/CFzr/Cdh1 activity
(Fig. 7). Our data support this
hypothesis. First, we find that the periodicity of Orc1 levels during the
endocycle requires a functional O-box, consistent with the cyclic destruction
of Orc1 by APC/CFzr/Cdh1 (Araki
et al., 2003; Araki et al.,
2005). Second, the levels of Orc1 are sensitive to Cyclin E.
Specifically, overexpressing Cyclin E after cells have entered the endocycle
results in the accumulation of APC/CFzr/Cdh1 targets, including
Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship
observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during
mitotic cycles is conserved during endocycles
(Vidwans et al., 2002).
Finally, in endocycling cells the accumulation of Orc1 occurs during periods
of high Cyclin E/Cdk2 activity, when APC/CFzr/Cdh1 dependent
proteolysis would be predicted to be low. These data support the idea that the
oscillations of Cyclin E/Cdk2 activity drive the periodicity of
APC/CFzr/Cdh1 activity during the endocycle.
Although we note that we have not formally demonstrated a requirement for
the oscillation of APC/CFzr/Cdh1 activity during the
Drosophila endocycle, it is interesting to speculate on how the
cyclic, rather than the continuous, activity of the APC/C might serve to
facilitate endocycle progression. Our data indicate that a period of high
APC/CFzr/Cdh1 activity is required during the G phase of the
endocycle in order to degrade the mitotic cyclins and Geminin, which can
function to inhibit the formation of pre-RCs. However, a period of low APC/C
activity may also be important. The continuous activation of
APC/CCdh1 significantly slows DNA replication in mouse tissue
culture cells (Sorensen et al.,
2000). This inhibition may reflect the inability of a cell to
accumulate adequate levels of proteins required for DNA replication, such as
the APC/CCdh1 target and pre-replication complex component CDC6, in
the presence of a constitutively active APC/CCdh1. In
Drosophila, continuous APC/CFzr/Cdh1 activity might
prevent the accumulation of two pre-RC components, CDC6 and Orc1.
Intriguingly, APC/C activity also appears to oscillate during mammalian
endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A
oscillate, consistent with the regulated destruction of the Cyclin A protein
by the APC/C (MacAuley et al.,
1998). Additionally, the inhibition of APC/C activity in
endocycling trophoblasts results in the accumulation of the APC/C targets
Cyclin A and Geminin (Gonzalez et al.,
2006). Taken together, these observations support a model in which
the oscillation of APC/CFzr/Cdh1 activity, which is driven by the
regulatory influences of Cdks, promotes efficient cell cycle progression
during the endocycle.
Our data raises important questions. Why do levels of some
APC/CFzr/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin,
remain below the level of detection while the levels of Orc1 protein
oscillate? What might account for these different modes of regulation?
Currently, there is no definitive explanation. However, we envisage at least
three possibilities, which are not mutually exclusive, that may contribute to
this differential behavior. First, we find that relative to the Cyclin
A and geminin, the levels of Orc1 transcript are only
minimally downregulated upon entry into the endocycle (see Fig. S4 in the
supplementary material). Transcriptional downregulation, or changes in
transcript stability, may help contribute to the low levels of Geminin and
Cyclin A proteins observed during the endocycle. Second, the translational
efficiency of a subset of transcripts may be reduced upon entry into the
endocycle. Finally, it is possible that the Orc1 protein is not as efficiently
targeted by the APC/CFzr/Cdh1 as the mitotic cyclins or Geminin.
Indeed the cis-acting sequences that target these proteins for destruction
show considerable variability. Orc1 is targeted for APC/CFzr/Cdh1
destruction via a novel motif called the O-box
(Araki et al., 2005). By
contrast, Cyclin B and Geminin are targeted by a similar but unique sequence
called the destruction-box (D-box), while Drosophila Cyclin A is
targeted for destruction by a large complex N-terminal degradation sequence
(Jacobs et al., 2001;
McGarry and Kirschner, 1998).
There is precedence for post-translational regulation of
APC/CFzr/Cdh1 targets, resulting in differential expression. In
mammalian cells the pre-RC component CDC6, which is structurally related to
Orc1, is protected from APC/CFzr/Cdh1 degradation by
phosphorylation by Cyclin E/Cdk2 (Laronne
et al., 2003; Mailand and
Diffley, 2005). One or all of these potential mechanisms may
contribute to the differential expression of various APC/CFzr/Cdh1
targets during the endocycle.
Recent evidence from mice indicates that the depletion of the APC/C
inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs,
such as geminin and Cyclin A, as well as APC/C activation
(Verschuren et al., 2007). The
authors suggest that the regulation of APC/C activity, by the inhibitor
Emi1/Rca1, drives a positive feedback circuit that controls both protein
stability and mRNA expression. Thus, the observed decrease in the levels of at
least some APC/C targets that occurs upon depletion of Emi1/Rca1, including
Geminin and Cyclin A, are controlled at the levels of transcription and
protein stability (Verschuren et al.,
2007). Developmentally programmed endocycles may provide a natural
example where cell cycle progression occurs in the context of increased
APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit
may be operating during Drosophila endocycles to downregulate the
transcription of E2F target genes. Determining the precise regulatory
relationships between the upregulation of APC/CFzr/Cdh1 activity
and the transcriptional downregulation of genes such as Cyclin A and
geminin, during the Drosophila endocycle represents an
exciting area for future research.
The requirement for APC/C activity to promote endocycle progression may
help answer several longstanding questions concerning the regulation of the
Drosophila endocycle. For example, why does the continuous expression
of Cyclin E inhibit cell cycle progression during the endocycle but not the
mitotic cycle (Edgar and Orr-Weaver,
2001; Follette et al.,
1998; Weiss et al.,
1998)? Several models have been proposed to explain this
difference. First, the breakdown of the nuclear envelope that occurs during
the mitotic cycle, but not the endocycle, may allow for a transient decrease
in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA
replication origins (Edgar and Orr-Weaver,
2001). Alternatively, there may be differences in the machinery
required to produce a functional pre-RC in mitotic versus endocycling cells
(Feger et al., 1995;
Lake et al., 2007). Our
results suggest an alternative model for why endocycles are unusually
sensitive to continuous Cyclin E expression. This model is based on our
demonstration that endocycle progression requires APC/C activity. Both
Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C
(Dawson et al., 1995;
Sigrist et al., 1995;
Sigrist and Lehner, 1997).
However, the regulation of these APC/C activators is very distinct
(Thornton and Toczyski, 2006).
During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent
on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1
(Rudner and Murray, 2000;
Shteinberg and Hershko, 1999;
Shteinberg et al., 1999;
Yamada et al., 1997). By
contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates
APC/CFzr/Cdh1 activity to late M phase and G1. Because of its
requirement for Cdk1 activity, APC/CFzy/Cdc20 is unlikely to be
active during most endocycles. Indeed, Drosophila endocycles proceed
normally in fzy mutants (Sigrist
et al., 1995). Thus, the only available activator of the APC/C
during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is
inhibited by Cyclin E/Cdk2 activity
(Sigrist and Lehner, 1997).
Therefore, we propose that during the endocycle, continuous Cyclin E/Cdk2
activity results in the permanent inhibition of the only available activator
of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A
and other potential targets, which act to block cell cycle progression. Thus,
the ability of continuous Cyclin E to inhibit DNA replication during the
endocycle may reflect differences in the available activators of the APC/C
present in mitotic versus endocycling cells.
In conclusion, our demonstration that the APC/CFzr/Cdh1 has a crucial function during endocycles will allow new models on the minimum cell cycle inputs necessary to construct a G/S oscillator to be formulated and tested.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1451/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
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