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First published online 19 July 2006
doi: 10.1242/dev.02488
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BZMB, Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany.
* Author for correspondence (e-mail: chle{at}uni-bayreuth.de)
Accepted 12 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cyclin A, Fzr/Cdh1, String/Cdc25, Mitosis, G1 phase, Cell proliferation arrest
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Peters, 2002).
Metazoan species express three different types of mitotic cyclins: A, B and
B3. The specific functions of these different cyclins are not understood in
detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin
B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their
functional specificity in Drosophila melanogaster. In this organism,
development to the adult stage requires the zygotic function of CycA,
but not of CycB or CycB3
(Jacobs et al., 1998;
Lehner and O'Farrell, 1989).
Initial analysis of the embryonic cell proliferation program in CycA
mutants revealed that epidermal cells fail to progress through the sixteenth
round of mitosis (Lehner and O'Farrell,
1989; Knoblich and Lehner,
1993). Cyclin A is also required for mitosis 16 in the epidermis
of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer
dependent upon completion of the preceding S phase
(Jacobs et al., 2001). The
failure of mitosis 16 in CycA mutants therefore does not simply
result from the activation of a DNA replication or damage checkpoint - a
possibility suggested by evidence obtained in vertebrate cells in which Cyclin
A binds not only to Cdk1 but also to Cdk2, and provides crucial functions
during S phase (Coverley et al.,
2002; Machida et al.,
2005; Pagano et al.,
1992).
The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also
occurs in CycA mutants (Lehner
and O'Farrell, 1990) (see below), complicates the explanation of
why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg
extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A
(Murray and Kirschner, 1989).
Conversely, mitosis is clearly inhibited in cultured human cells after the
microinjection of antibodies against cyclin A
(Pagano et al., 1992). Cyclin
A-Cdk1 complexes are thought to have special properties, important for
starting up a positive-feedback loop that confers a switch-like behavior on
the Cdk1 activation process (Clarke et al.,
1992; Pomerening et al.,
2005). In this feedback loop, Cdk1 activity results in
phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in
phosphorylation and activation of the String/Cdc25 phosphatase, which removes
the inhibitory phosphate modifications from Cdk1. However, our analyses
indicate that the Cyclin A requirement in Drosophila is not linked to
this positive-feedback loop. Rather, it is linked to the fact that the
sixteenth round of mitosis during embryogenesis is the last cell division for
the great majority of the epidermal cells.
After mitosis 16, most epidermal cells enter a G1 phase and become
mitotically quiescent (Edgar and O'Farrell,
1990; Knoblich et al.,
1994). By contrast, all the previous embryonic divisions (mitoses
1-15) are followed by an immediate onset of S phase. The G1 phase after
mitosis 16 is therefore the first G1 phase during development. Entry into this
G1 phase is dependent upon a complete, developmentally controlled inactivation
of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry
into S phase (Knoblich et al.,
1994; Sprenger et al.,
1997). Cyclin E-Cdk2 inactivation results from transcriptional
CycE downregulation and concomitant upregulation of dacapo,
which encodes the single Drosophila CIP/KIP-type inhibitor specific
for Cyclin E-Cdk2 (de Nooij et al.,
1996; Knoblich et al.,
1994; Lane et al.,
1996). Cyclin A-Cdk1 inactivation is dependent on Fzr (Rap -
FlyBase), which is also transcriptionally upregulated. Moreover, Fzr is
activated as a consequence of Cyclin E-Cdk2 inactivation
(Sigrist and Lehner, 1997).
Importantly, this cell cycle exit program is initiated already during G2 of
the final division cycle.
Although cycle 16 is the final division cycle for most epidermal cells,
some defined regions do not activate the cell cycle exit program during cycle
16 (Knoblich et al., 1994).
Instead, they maintain CycE expression, enter S phase immediately
after mitosis 16 and complete an additional division cycle 17. In these
regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A
is therefore especially important for terminal mitoses preceding G1 and cell
cycle exit. We show that the downregulation of Cyclin E-Cdk2 before terminal
divisions, in preparation for the imminent cell cycle exit, converts Cyclin A
from a redundant into an indispensable, negative regulator of
Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers
String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle
regulator Cyclin A therefore depends on the developmental context.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Lehner et al.,
1991), UAS-CycA
(Weiss et al., 1998),
CycEAR95 (Knoblich et
al., 1994), dap4 and UAS-CycE
(Lane et al., 1996),
fzrie28 and fzrG0326
(Jacobs et al., 2002),
UAS-fzr (Sigrist and Lehner,
1997), Hs-CycB, Hs-
CycB, Hs-CycB3 and
Hs-CycB3 (Sigrist
et al., 1995), Hs-stg
(Edgar and O'Farrell, 1990),
Hs-Cdk1 (Stern et al.,
1993), Hs-Cdk1AF
(Sprenger et al., 1997) and
prd-GAL4 (Brand and Perrimon,
1993). UAS-fzrpsm lines were generated by
standard germ line transformation. The CycA, CycE and fzr
alleles were kept over the blue balancer chromosomes TM3, Sb1
P{35UZ}2, CyO, P{ftz/lacB}E3 and FM7i, P{ftz/lacC}YH1,
respectively. The characteristic lacZ expression patterns driven by
the transgenes on these blue balancers allowed embryo genotype identification
after anti-ß-galactosidase immunolabeling. As all transgenes were
established in a w1 background, we used this strain for
some control experiments.
Construction of the UAS-fzrpsm transgene
To generate UAS-fzrpsm lines, we first changed the
codons for the serines at positions 3, 15, 60, 121, 136, 148, 211, 307 and
437, and for the threonines at positions 30, 109 and 285 in a fzr
cDNA into alanine codons using site-directed mutagenesis (QuikChange,
Stratagene). Moreover, the codon for serine 349 was changed into a cysteine
codon, which is found at the corresponding position in Drosophila,
Xenopus and budding yeast Fzy/Cdc20. The mutated cDNA was subsequently
inserted into the EcoRI and BglII sites of pUAST
(Brand and Perrimon, 1993). All
mutations were verified by DNA sequence analysis.
Bromodeoxyuridine pulse labeling and immunolabeling
Pulse labeling of embryos (6.5-7.5 hours after egg deposition) with
5'-bromodeoxyuridine (BrdU) for 20 minutes and immunolabeling with
antibodies specific for BrdU (Becton-Dickinson), ß-galactosidase
(ICN/Cappel and Promega), 
-tubulin (Sigma), phospho-histone H3 (Upstate
Biotechnology), Cyclin E (Lane et al.,
1996), Cyclin B and Cyclin B3
(Jacobs et al., 1998), and with
the DNA stain Hoechst 33258, were carried out essentially as described
previously (Lehner et al.,
1991). For secondary antibodies, we used goat antibodies
conjugated with Cy3 (Jackson ImmunoResearch) or Alexa Fluor 488 (Molecular
Probes). Images were captured using cooled CCD cameras and processed using
PhotoShop (Adobe).
Temperature shifts
Heat shocks were applied as described previously
(Sigrist et al., 1995). Eggs
were collected for 1 hour and aged for 5.5 hours at 25°C before the
application of a heat shock (30 minutes, 37°C). After a recovery period at
25°C of either 30 minutes (data not shown) or 120 minutes
(Fig. 2), embryos were
fixed.
Immunoblotting
For determination of String/Cdc25 phosphatase levels in fzr mutant
embryos, eggs were collected from fly stocks with either
fzrie28 or fzrG0326 over the FM7i,
P{ftz/lacC}YH1 blue balancer chromosome for 2 hours and aged for 8 hours
at 25°C. Hemi- or homozygous fzrie28 and
fzrG0326 embryos were sorted from sibling blue balancer
embryos after methanol fixation and anti-ß-galactosidase immunolabeling,
as described previously (Jacobs et al.,
2002). Moreover, for control experiments, eggs were also collected
from w1 and Hs-stg fly stocks for 2 hours. After
ageing for 8 hours at 25°C, collections were divided into two aliquots.
One of these was exposed to a heat shock (15 minutes, 37°C) and allowed to
recover (15 minutes, 25°C), whereas the other was kept at 25°C. Embryo
extract preparation and immunoblotting was done as described
(Knoblich et al., 1994).
Antibodies against String (Edgar et al.,
1994) and -tubulin (Sigma) were used for detection.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lehner et al., 1991).
Interestingly, these mitotic cells were not distributed in a random pattern
within the epidermis. Instead, they were found within defined regions where
epidermal cells progress through a seventeenth division cycle instead of
becoming quiescent after mitosis 16. The largest of these domains corresponds
to the prospective anterior spiracle region situated mostly in the first, and
to a minor extent in the second, thoracic segment. In wild-type embryos, this
epidermal region is efficiently labeled during a BrdU pulse after mitosis 16,
in contrast to the adjacent postmitotic epidermal cells
(Fig. 1E,F) (see also
Knoblich et al., 1994;
Sauer et al., 1995).
Quantitative analyses of mitotic cells at the stage of mitosis 16 in embryos
with various strong or null alleles of CycA clearly revealed that 80%
of the mitotic cells in the epidermis were confined to the prospective
anterior spiracle region in the mutants
(Fig. 1C,D,K). By contrast,
mitotic cells were almost evenly distributed over the thoracic and abdominal
segments, and were not confined to the prospective anterior spiracle region in
CycA+ control embryos
(Fig. 1A,B,K). The pattern of
mitotic cells in CycA mutants at the stage of mitosis 16 indicates
therefore that zygotic CycA+ expression is primarily
required for entry into mitosis in those cells that enter G1/0 and exit from
the cell cycle after mitosis 16. By contrast, mitosis is not absolutely
dependent on zygotic CycA+ function in cells that continue
to proliferate after mitosis 16.
We emphasize that not all of the epidermal cells in the anterior spiracle
region progress normally through mitosis 16 in CycA mutants. Previous
analyses have clearly demonstrated that a fraction of these cells fail to
divide and yet continue with an additional round of DNA replication
(Sauer et al., 1995). At the
stage of mitosis 16, therefore, the requirement for zygotic CycA
expression is maximal for terminal mitoses and variable for non-terminal
mitoses. Some of the latter mitoses appear to be normal, and only restricted
aspects of mitosis like the re-licensing of DNA replication are realized in
others.
The notion that Cyclin A is especially crucial for terminal divisions is
further supported by observations in the nervous system. Lear et al.
(Lear et al., 1999) have
reported that the divisions of ganglion mother cells in various neuroblast
lineages are preferentially inhibited in CycA mutants. Ganglion
mother cell divisions are terminal mitoses. The resulting daughter cells exit
from the cell cycle and differentiate into post-mitotic neurons. By contrast,
the asymmetric neuroblast divisions, which were found to be less inhibited in
CycA mutants, are not terminal mitoses. The resulting daughter cells,
a neuroblast and a ganglion mother cell, progress through additional divisions
in the wild type. Consistent with these findings, mitotic cells (including
normal anaphase figures) were readily observed in the nervous system of
CycA mutant embryos during the later stages 12-14
(Fig. 1I,J). BrdU incorporation
has previously been observed in the central nervous system of CycA
mutants during these late stages (Lehner
et al., 1991). We conclude, therefore, that at least some cells
continue to progress through apparently normal division cycles in
CycA mutants embryos, even at late stages, long after the depletion
of the maternal contribution (Knoblich and
Lehner, 1993) and long after the failure of the terminal mitosis
16 in the epidermis.
|
). Cyclin
A-Cdk1 activity might therefore start the positive Cdk1-activation loop,
involving the inhibition of Wee1 kinase and activation of the Cdk1 activator
String/Cdc25 phosphatase. Although Cyclin A-Cdk1 complexes in
Drosophila embryos are subject to inhibitory phosphorylation
(Sprenger et al., 1997), it is
conceivable that the responsible kinases in this organism also work less
efficiently on Cyclin A-Cdk1 complexes than on Cdk1 complexes containing
B-type cyclins. Accordingly, epidermal cells of CycA mutant embryos
might fail to enter mitosis 16 because of an inability to start up the
positive Cdk1-activation loop. In this case, expression of a mutant Cdk1
(Cdk1AF) (Sprenger et al.,
1997), which cannot be phosphorylated by Wee1/Myt1-like kinases,
should restore entry into mitosis 16 in the epidermis of CycA
mutants. For an experimental test, we crossed a transgene that allows the
heat-inducible expression of Cdk1AF into CycA mutants.
However, expression of this transgene did not restore mitosis 16 in the mutant
epidermis (Fig. 2A-L). This
observation also suggested that absence of String/Cdc25 activity is not the
only cause for the failure of mitosis 16 in the mutants and raised the
possibility that other Cdk1 activators might be lacking.
Therefore, we carefully re-evaluated the behavior of Cyclin B and Cyclin B3
in CycA mutants. Our previous analysis of CycA mutants had
clearly demonstrated that Cyclin B re-accumulates in the embryonic epidermis
after degradation during mitosis 15 and eventually disappeared again late in
cycle 16 (Lehner and O'Farrell,
1990). The precise timing of this disappearance was difficult to
assess with fixed CycA mutants because it occurs during a phase with
little morphological change. In addition, mitotic divisions, which normally
allow for precise staging because of the dynamic but stereotypic nature of the
division program during wild-type embryogenesis, do not accompany this
disappearance of Cyclin B in CycA mutants. However, by crossing
prd-GAL4 and UAS-CycA into CycA mutants, embryos
with segmentally alternating regions either with or without zygotic
CycA expression can be generated
(Jacobs et al., 2001). The
CycA-expressing segments in these embryos progress normally through
the wild-type division program. By contrast, the intervening CycA
mutant segments fail to progress through the terminal mitosis 16 in the
epidermis. Immunolabeling of such embryos demonstrated that Cyclin B3
disappeared rapidly from the epidermal regions lacking Cyclin A just before
the stage where entry into terminal mitosis 16 normally occurs in the
neighboring CycA-expressing segments
(Fig. 3). Immunolabeling with
antibodies against Cyclin B revealed an analogous premature disappearance
before terminal mitoses (data not shown)
(Dienemann and Sprenger, 2004).
Interestingly, premature disappearance was not observed within the prospective
anterior spiracle region of CycA mutant embryos where mitosis 16 is
not the terminal mitosis (Fig.
3A, arrow).
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Therefore, we addressed whether String/Cdc25 phosphatase might disappear
prematurely in CycA mutants before mitosis 16, as described above for
Cyclin B and Cyclin B3. Previous observations have strongly suggested that
String/Cdc25, which includes a KEN box, is also an APC/C substrate
(Edgar and Datar, 1996;
Edgar et al., 1994). If absence
of Cyclin A was to cause a premature APC/C activation before terminal mitoses,
String/Cdc25 might be eliminated along with the mitotic cyclins. Our
comparison of String/Cdc25 levels in wild-type and fzr mutant embryos
by immunoblotting experiments supported the idea that String/Cdc25 is an APC/C
substrate (Fig. 4). In
fzr mutants, String/Cdc25 levels were found to be elevated about
tenfold. Unfortunately, the available antibodies against String/Cdc25 did not
work in immunolabeling experiments with CycA mutant embryos
expressing prd-Gal4 and UAS-CycA. However, as an
experimental test for the hypothesis that the failure of entry into the
terminal mitosis 16 in the epidermis of CycA mutant embryos reflects
the premature combined degradation of B-type cyclins and String/Cdc25, we
simultaneously induced three heat-inducible transgenes in CycA mutant
embryos, resulting in the expression of a String/Cdc25-independent Cdk1
(Cdk1AF) along with N-terminally truncated Cyclins B and B3 lacking
destruction boxes. Expression of these three transgenes fully restored entry
into mitosis 16 (Fig. 2M-P).
After entry into mitosis, epidermal cells were unable to complete this
division because of the presence of the non-degradable B-type cyclins, as
expected (Sigrist et al.,
1995).
Simultaneous overexpression from analogous heat-inducible transgenes of wild-type Cdk1 in combination with the two non-degradable B-type cyclins did not restore mitosis 16 in the epidermis of CycA mutants (Fig. 2Q-T). The same result was also obtained with combinations of non-degradable B-type cyclins and wild-type String/Cdc25 (Fig. 2U,V), or wild-type B-type cyclins and Cdk1AF (Fig. 2W,X). Finally, the combination of wild-type String/Cdc25 and Cyclin B and Cyclin B3 with destruction boxes did not restore mitosis 16 either (data not shown). These results indicate therefore that the failure of terminal mitosis 16 in CycA mutant embryos results from the combined loss of B-type cyclins and String/Cdc25. Moreover, the instability of these mitotic inducers before the terminal mitosis 16 in CycA mutant embryos appears to be so extensive that it cannot be overcome by the overexpression of wild-type Cdk1 activator proteins with functional degradation signals.
Terminal mitosis 16 in CycA mutants is restored by the loss of Fzr or gain of Cyclin E
The loss of String/Cdc25 activity and B-type cyclins before the terminal
mitosis in the epidermis of CycA mutant embryos might reflect a
premature activation of Fzr activity. Fzr has been shown to be required for
the degradation of mitotic cyclins during the G1 phase, when the epidermal
cells exit from the division cycle after mitosis 16
(Sigrist and Lehner, 1997).
The level of fzr transcripts is upregulated during wild-type
embryogenesis when cells exit from the division cycle
(Sigrist and Lehner, 1997).
Moreover, in parallel with fzr upregulation, CycE, which
acts as a negative regulator of Fzr, is downregulated
(Knoblich et al., 1994).
Because these changes are initiated already before mitosis 16, but only in
epidermal cells programmed to exit from the cell cycle after mitosis 16, we
investigated the possibility that the onset of this cell cycle exit program
might create the higher Cyclin A requirement during terminal mitoses.
To evaluate whether the premature loss of String/Cdc25 activity and B-type
cyclins that is responsible for the failure of the terminal mitosis in the
epidermis of CycA mutants is brought about by Fzr, we analyzed
fzr CycA double mutant embryos. In these embryos, mitosis 16 was
fully restored in the normal pattern (Fig.
5A-D) (Sigrist and Lehner,
1997) (and data not shown), and premature B-type cyclin
degradation before mitosis 16 no longer occurred. Moreover, the epidermal
cells in these double mutant embryos even progressed through an additional
seventeenth division cycle accompanied by B-type cyclin accumulation and
degradation during interphase and mitosis, respectively
(Fig. 5G), as was also observed
in fzr single mutant embryos (Fig.
5F) (Jacobs et al.,
2002; Sigrist and Lehner,
1997). Although not all of the late mitotic figures in fzr
CycA double mutant embryos were normal, most epidermal cells appeared to
progress successfully through mitosis 17, as was also observed in the
fzr single mutants (Fig.
5H,I). These results indicate that the Cyclin A requirement for
mitosis is minimal in the absence of Fzr. Moreover, they suggest that a
premature Fzr activation is responsible for the loss of String/Cdc25 and
B-type cyclins that prevents entry into the terminal mitosis 16 in the
epidermis of CycA mutants.
|
). This
extended UAS-CycE expression fully restored mitosis 16 in
CycA mutant embryos (Fig.
6), suggesting that the downregulation of endogenous CycE
expression indeed contributes to the arrest before the terminal mitosis
observed in the epidermis of CycA mutants. As expected, we also no
longer observed premature Cyclin B3 degradation in the
UAS-CycE-expressing segments of the CycA mutants
(Fig. 6A,B).
prd-GAL4-driven UAS-CycE expression not only restored
progression through mitosis 16 in CycA mutants, but also promoted
progression through an extra S phase 17 (data not shown), as was observed in
wild type (Knoblich et al.,
1994).
Cyclin E and Cyclin A are both negative regulators of Fzr
Although the elimination of fzr function in CycA mutants
restored the terminal mitosis 16, fzr overexpression is known to
phenocopy CycA mutants. In segments with prd-GAL4-driven
UAS-fzr expression, mitotic cyclins disappear prematurely just before
the stage of mitosis 16, and this terminal division is inhibited
(Fig. 7C,D)
(Sigrist and Lehner, 1997).
This premature fzr activation resulting from UAS-fzr
overexpression is suppressed by UAS-CycE co-expression
(Sigrist and Lehner, 1997).
The premature fzr activation apparent in CycA mutants
suggested that Cyclin A might also function as a negative Fzr regulator, like
Cyclin E. In addition, a redundant ability of Cyclin A and E to suppress
mitotic cyclin degradation during G2 has also been suggested by previous
studies (Sprenger et al.,
1997; Vidwans et al.,
2002). Accordingly, co-expression of UAS-CycA should also
suppress the UAS-fzr overexpression phenotype, as previously
described for UAS-CycE. This was found to be the case
(Fig. 7E,F).
In addition, an analysis of CycA CycE double mutant embryos also
provided evidence consistent with the notion that Cyclin A and E might
co-operate as negative Fzr regulators. In these double mutant embryos, cells
were observed to arrest before mitosis 15
(Fig. 8), whereas this mitosis
is not affected in single mutants. BrdU pulse labeling did not reveal
abnormalities during the preceding S phase 15 in the double mutants (data not
shown), arguing against the possibility that the arrest before mitosis 15
reflects the activation of a DNA replication check point. However,
immunolabeling with antibodies against Cyclin B (data not shown) or Cyclin B3
(Fig. 8B) indicated that these
mitotic cyclins had disappeared already around the stage of mitosis 15 in
CycA CycE double mutants and thus even earlier than in CycA
single mutants. Low levels of Fzr, mostly maternally derived, which are
present during cycle 15 (Raff et al.,
2002), therefore appear to become activated efficiently in
CycA CycE double mutants. All of our observations therefore support
the notion that both Cyclin A and Cyclin E act as negative Fzr regulators.
The Drosophila CIP/KIP-type Cdk inhibitor Dacapo specifically
inhibits Cyclin E-Cdk2 (de Nooij et al.,
1996; Lane et al.,
1996). dapcapo (dap) expression is known to be
upregulated during the final division cycle in the embryonic epidermis
(de Nooij et al., 1996;
Lane et al., 1996). Therefore,
we analyzed whether genetic elimination of dap function restores
progression through the final epidermal mitosis in CycA mutants.
However, mitosis 16 was not restored in dap, CycA double mutants
(Fig. 8N, data not shown).
Interestingly, the epidermal cells in dap, CycA double mutants
entered another S phase after the stage of mitosis 16
(Fig. 8N). It appears therefore
that the inhibition of Cyclin E-Cdk2 activity after dap upregulation
is not effective before mitosis 16. However, Dap is required thereafter to
prevent Cyclin E-Cdk2 from inducing entry into S phase after origin
re-licensing during exit from mitosis 16 in wild-type embryos
(Fig. 8M)
(de Nooij et al., 1996;
Lane et al., 1996), or
resulting from premature Fzr activation in CycA mutants
(Fig. 8N).
In both budding yeast and human cells, phosphorylation of Fzr/Cdh1 by Cdks
has an inhibitory effect (Kramer et al.,
2000; Lukas et al.,
1999; Sorensen et al.,
2001; Zachariae et al.,
1998). In these organisms, Cdh1 proteins with mutations in Cdk
consensus phosphorylation sites were shown to be hyperactive and resistant
against inhibition by Cdk activity (Lukas
et al., 1999; Sorensen et al.,
2001; Zachariae et al.,
1998). Interestingly, however, Cdk2 has been shown to inhibit Cdh1
in human cells only in conjunction with Cyclin A, and not with Cyclin E
(Lukas et al., 1999). In an
attempt to address the mechanisms whereby Cyclin A and Cyclin E inhibit Fzr in
Drosophila embryos, we also mutated all of the thirteen Cdk consensus
phosphorylation sites in Fzr (all positions with S or T followed by P) and
analyzed its function. prd-GAL4-mediated expression of
UAS-fzrpsm (psm: phosphorylation sites mutated) resulted
in the same phenotype that had been previously observed with UAS-fzr
wild type, indicating that the Fzrpsm protein is still functional
(compare Fig. 7C,D with 7G,H).
Unexpectedly, however, this UAS-fzrpsm phenotype was still
suppressed by co-expression of either UAS-CycE
(Fig. 7I,J) or
UAS-CycA (Fig. 7K,L),
in contrast to the findings in human cells. It is possible, therefore, that
Cyclin E and Cyclin A inactivate Fzr by phosphorylation of non-consensus sites
or that these cyclins might act on additional targets to inhibit
Fzr-APC/C-mediated degradation.
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|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), null
mutations in CycA result in embryonic lethality
(Lehner and O'Farrell, 1989).
Here, we have clarified the molecular basis of the distinct importance of
Cyclin A. Our results indicate that the crucial role of Cyclin A is linked to
its ability to inhibit Fzr-APC/C-mediated degradation. Moreover, we show that
this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation
pathway is of particular importance for progression through the very last
mitotic division preceding cell cycle exit and the proliferative quiescence of
epidermal cells during embryogenesis. This particular Cyclin A requirement
during terminal divisions is caused by a cell cycle exit program that is
initiated already before the terminal mitosis. The cell cycle exit program
includes downregulation of Cyclin E-Cdk2, which has a comparable ability to
inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of
Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an
indispensable inhibitor of the premature degradation of mitotic cyclins and
String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, we have
shown here that the terminal mitosis in the epidermis of CycA mutants
can be restored by overexpression of Cyclin E, by genetic elimination of Fzr,
or by simultaneous expression of the String/Cdc25-independent
Cdk1AF mutant and B-type cyclin versions that are no longer
Fzr-APC/C substrates.
|
;
Sigrist and Lehner, 1997). Two
findings, however, strongly suggest that Cyclin A functions not just
downstream of Fzr, but also upstream as a negative regulator. The observed
premature loss of B-type cyclins in CycA mutants is readily explained
by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to
explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression
of the UAS-fzr overexpression phenotype by co-expression of
UAS-CycA, which we describe here, includes the re-accumulation of
B-type cyclins and not just the restoration of terminal mitosis 16.
Indirect regulation of Fzr by Cyclin A and Cyclin E
Work in mammalian cells has clearly established that Cyclin A functions as
a negative regulator of Fzr/Cdh1 (Lukas et
al., 1999; Sorensen et al.,
2001). Human Cyclin A can bind directly to Cdh1
(Lukas et al., 1999;
Sorensen et al., 2001).
Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in
the dissociation of Cdh1 from APC/C (Lukas
et al., 1999; Sorensen et al.,
2001). Conversely, mutations in Cdk consensus phosphorylation
sites of human CDH1 were reported to abolish inhibition by Cyclin A
(Lukas et al., 1999). Our
findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Our
Fzrpsm variant no longer contains canonical Cdk consensus
phosphorylation sites (S/T P) and yet its activity is still suppressed by
CycA overexpression. Fzr inhibition by CyclinA-dependent
phosphorylation of non-consensus sites remains a possibility in
Drosophila. However, we point out that, apart from a potential
control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like
Drosophila protein Rca1 (Dienemann
and Sprenger, 2004). Rca1 overexpression has been shown to prevent
premature Cyclin B degradation and restore mitosis 16 in the epidermis of
CycA mutant embryos (Dienemann and
Sprenger, 2004). Based on these observations, the failure of
mitosis 16 in CycA mutants was proposed to reflect premature Fzr
activation (Dienemann and Sprenger,
2004), a suggestion fully confirmed by our work. It is
conceivable, therefore, that the Cyclin A-mediated suppression of
Fzrpsm activity involves Rca1 or other unknown targets. The fact
that not only Cyclin A, but also Cyclin E, effectively suppresses
Drosophila Fzr and Fzrpsm provides further support of
additional regulatory complexity. In vertebrate systems, only Cyclin A and not
Cyclin E was shown to bind and inhibit Cdh1
(Lukas et al., 1999).
|
|
;
Lane et al., 1996), apart from
the downregulation of Cyclin E (Knoblich
et al., 1994) and the upregulation of Fzr
(Sigrist and Lehner, 1997).
Accordingly, genetic elimination of dacapo function should also
restore progression through terminal mitosis 16 in CycA mutants.
However, mitosis 16 was not observed in the epidermis of dacapo CycA
double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears
to be insignificant before mitosis 16. After the stage of mitosis 16, however,
the epidermal cells in these double mutants entered an endoreduplication
cycle, a behavior that is also displayed by some cells in the prospective
anterior spiracle region of CycA single mutants
(Sauer et al., 1995). This
region does not downregulate Cyclin E during cycle 16 in the wild type, it
does not upregulate Dacapo, and it progresses through an additional cycle 17
instead of becoming postmitotic after mitosis 16, in contrast to the great
majority of the other epidermal cells. The premature activation of Fzr in
CycA mutants, therefore, appears to result in DNA replication origin
re-licensing, perhaps as a result of B-type cyclin and geminin degradation.
Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells
in which it is not effectively eliminated by both Cyclin E downregulation and
Dacapo upregulation. Importantly, not all cells in the anterior spiracle
region of CycA mutants endoreduplicate, some of the cells still
manage to divide. This variability could reflect minor differences in the
onset and strength of the zygotic Cyclin E expression. The outcome of
insufficient Cyclin A levels appears to be highly dependent on the levels of
Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in
particular during cell cycle exit. The significance of basic cell cycle
regulators in vivo is therefore different in various tissues and developmental
stages, and most likely in various cultured mammalian cell types as well. |
ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118,401 -415.[Abstract]
Clarke, P. R., Leiss, D., Pagano, M. and Karsenti, E. (1992). Cyclin A-dependent and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts. EMBO J. 11,1751 -1761.[Medline]
Coverley, D., Laman, H. and Laskey, R. A. (2002). Distinct roles for cyclins E and A during DNA replication complex assembly and activation. Nat. Cell Biol. 4, 523-528.[CrossRef][Medline]
de Nooij, J. C., Letendre, M. A. and Hariharan, I. K. (1996). A cyclin-dependent kinase inhibitor, dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 87,1237 -1247.[CrossRef][Medline]
Dienemann, A. and Sprenger, F. (2004). Requirements of cyclin A for mitosis are independent of its subcellular localization. Curr. Biol. 14,1117 -1123.[CrossRef][Medline]
Edgar, B. A. and O'Farrell, P. H. (1990). The three postblastoderm cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell 62,469 -480.[CrossRef][Medline]
Edgar, B. A. and Datar, S. A. (1996). Zygotic
degradation of two maternal Cdc25 mRNAs terminates Drosophila's early cell
cycle program. Genes Dev.
10,1966
-1977.
Edgar, B. A., Sprenger, F., Duronio, R. J., Leopold, P. and
O'Farrell, P. H. (1994). Distinct molecular mechanisms
regulate cell cycle timing at successive stages of Drosophila embryogenesis.
Genes Dev. 8,440
-452.
Jacobs, H. W., Knoblich, J. A. and Lehner, C. F.
(1998). Drosophila Cyclin B3 is required for female fertility and
is dispensable for mitosis like Cyclin B. Genes Dev.
12,3741
-3751.
Jacobs, H. W., Keidel, E. and Lehner, C. F. (2001). A complex degradation signal in Cyclin A required for G1 arrest, and a C-terminal region for mitosis. EMBO J. 20,2376 -2386.[CrossRef][Medline]
Jacobs, H. W., Richter, D. O., Venkatesh, T. R. and Lehner, C. F. (2002). Completion of mitosis requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog. Curr. Biol. 12,1435 -1441.[CrossRef][Medline]
Knoblich, J. A. and Lehner, C. F. (1993). Synergistic action of Drosophila cyclin A and cyclin B during the G2-M transition. EMBO J. 12,65 -74.[Medline]
Knoblich, J. A., Sauer, K., Jones, L., Richardson, H., Saint, R. and Lehner, C. F. (1994). Cyclin E controls S phase progression and its downregulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77,107 -120.[CrossRef][Medline]
Kramer, E. R., Scheuringer, N., Podtelejnikov, A. V., Mann, M.
and Peters, J. M. (2000). Mitotic regulation of the APC
activator proteins CDC20 and CDH1. Mol. Biol. Cell
11,1555
-1569.
Lane, M. E., Sauer, K., Wallace, K., Jan, Y. N., Lehner, C. F. and Vaessin, H. (1996). Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 87,1225 -1236.[CrossRef][Medline]
Lear, B. C., Skeath, J. B. and Patel, N. H. (1999). Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system. Mech. Dev. 88,207 -219.[CrossRef][Medline]
Lehner, C. F. and O'Farrell, P. H. (1989). Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell 56,957 -968.[CrossRef][Medline]
Lehner, C. F. and O'Farrell, P. H. (1990). The roles of Drosophila cyclin A and cyclin B in mitotic control. Cell 61,535 -547.[CrossRef][Medline]
Lehner, C. F., Yakubovich, N. and O'Farrell, P. H. (1991). Exploring the role of Drosophila cyclin A in the regulation of S-phase. Cold Spring Harb. Symp. Quant. Biol. 56,465 -475.[Medline]
Lukas, C., Sorensen, C. S., Kramer, E., Santoni-Rugiu, E., Lindeneg, C., Peters, J. M., Bartek, J. and Lukas, J. (1999). Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401,815 -818.[CrossRef][Medline]
Machida, Y. J., Hamlin, J. L. and Dutta, A. (2005). Right place, right time, and only once: replication initiation in metazoans. Cell 123, 13-24.[CrossRef][Medline]
Murray, A. W. (2004). Recycling the cell cycle: cyclins revisited. Cell 116,221 -234.[CrossRef][Medline]
Murray, A. W. and Kirschner, M. (1989). Cyclin synthesis drives the early embryonic cell cycle. Nature 339,275 -280.[CrossRef][Medline]
Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. and Draetta, G. (1992). Cyclin A is required at two points in the human cell cycle. EMBO J. 11,961 -971.[Medline]
Peters, J. M. (2002). The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9, 931-943.[CrossRef][Medline]
Pomerening, J. R., Kim, S. Y. and Ferrell, J. E., Jr (2005). Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations. Cell 122,565 -578.[CrossRef][Medline]
Raff, J. W., Jeffers, K. and Huang, J. Y.
(2002). The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the
destruction of cyclin B in space and time. J. Cell
Biol. 157,1139
-1149.
Rape, M., Reddy, S. K. and Kirschner, M. W. (2006). The processivity of multiubiquitination by the APC determines the order of substrate degradation. Cell 124,89 -103.[CrossRef][Medline]
Sauer, K., Knoblich, J. A., Richardson, H. and Lehner, C. F.
(1995). Distinct modes of cyclin E/cdc2c kinase regulation and S
phase control in mitotic and endoreduplication cycles of Drosophila
embryogenesis. Genes Dev.
9,1327
-1339.
Sigrist, S. J. and Lehner, C. F. (1997). Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. Cell 90,671 -681.[CrossRef][Medline]
Sigrist, S., Jacobs, H., Stratmann, R. and Lehner, C. F. (1995). Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3. EMBO J. 14,4827 -4838.[Medline]
Sorensen, C. S., Lukas, C., Kramer, E. R., Peters, J. M.,
Bartek, J. and Lukas, J. (2001). A conserved cyclin-binding
domain determines functional interplay between anaphase-promoting complex-Cdh1
and cyclin A-Cdk2 during cell cycle progression. Mol. Cell.
Biol. 21,3692
-3703.
Sprenger, F., Yakubovich, N. and O'Farrell, P. H. (1997). S phase function of Drosophila cyclin A and its downregulation in G1 phase. Curr. Biol. 7, 488-499.[CrossRef][Medline]
Stern, B., Ried, G., Clegg, N. J., Grigliatti, T. A. and Lehner, C. F. (1993). Genetic analysis of the Drosophila cdc2 homolog. Development 117,219 -232.[Abstract]
Vidwans, S. J., DiGregorio, P. J., Shermoen, A. W., Foat, B., Iwasa, J., Yakubovich, N. and O'Farrell, P. H. (2002). Sister chromatids fail to separate during an induced endoreplication cycle in Drosophila embryos. Curr. Biol. 12,829 -833.[CrossRef][Medline]
Weiss, A., Herzig, A., Jacobs, H. and Lehner, C. F. (1998). Continuous Cyclin E expression inhibits progression through endoreduplication cycles in Drosophila. Curr. Biol. 8,239 -242.[CrossRef][Medline]
Zachariae, W., Schwab, M., Nasmyth, K. and Seufert, W.
(1998). Control of cyclin ubiquitination by CDK-regulated binding
of Hct1 to the anaphase promoting complex. Science
282,1721
-1724.
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