1 Department of Molecular Pharmacology and Biological Chemistry, Robert H. Lurie
Comprehensive Cancer Center, Northwestern University, 303 East Chicago Avenue,
Chicago, IL 60611-3093, USA
2 Department of Molecular Biology and Genetics, Biotechnology Building, Cornell
University, Ithaca, NY 14853-2703, USA
* Author for correspondence (e-mail: andres{at}northwestern.edu )
Accepted 25 November 2001
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Summary |
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Key words: ida, APC5, Anaphase-promoting complex, Sister-chromatid separation, Cyclin B degradation, Mutant analysis, Drosophila melanogaster
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Introduction |
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The APC/cyclosome (APC/C) contains 8-13 subunits depending on species, and
functions as an E3 ubiquitin-protein ligase that marks specific substrates for
proteasome-dependent degradation (Holloway
et al., 1993). The APC/C generally controls the
metaphase-to-anaphase transition and mitotic exit during cell division.
Temporally regulated APC/C functions ensure that anaphase occurs only after
successful DNA replication, proper microtubule attachment to all chromosomes
via the kinetochore, and subsequent chromosome congression to the metaphase
plate (reviewed by Skibbens and Hieter,
1998
; Page and Hieter,
1999
).
Much of our current understanding of the function of the APC/C is based
primarily on biochemical and genetic work from fungal systems, including
Saccharomyces cerevisiae, Schizosaccharomyces pombe and
Aspergillus nidulans. Known functions of the APC/C complex include:
degradation of the Securin or sister-chromatid separation inhibitor proteins,
Pds1p and Cut2p (Cohen-Fix et al.,
1996; Funabiki et al.,
1997
); the regulated degradation of cyclins A, B and B3 during
prometaphase, metaphase and anaphase
(Sigrist et al., 1995
;
Parry and O'Farrell, 2001
);
and continued B-type cyclin degradation through G1 until the onset of S phase
(Amon et al., 1994
;
Irniger and Nasmyth, 1997
).
However, little is known about the biochemical function of individual APC/C
subunits.
Here we present the cloning, characterization, and mutant analysis of the
ida (imaginal discs arrested) gene.
ida encodes a homolog of the APC5 subunit
(Yu et al., 1998) of the APC/C
in Drosophila melanogaster. Our results are the first phenotypic
characterization of APC5 mutations in metazoans. As expected, our data suggest
that the APC5 subunit is required for late cell cycle events such as cyclin B
degradation. However, based on our cytological observations, we propose an
additional role for the APC5 subunit during chromosome congression, and
propose a model in which IDA controls regulatory subfunctions of the
APC/C.
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Materials and Methods |
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Cloning ida and characterizing mutant alleles
Total RNA (20 µg) from wild-type and ida107
third-instar larvae was used to generate northern blots using previously
described methods (Vaskova et al.,
2000
). Blots were hybridized with RNA probes synthesized against
the p63F.14, p63F.15 and p63F.16 subclones isolated from an
80 kb chromosomal walk of the 63F region
(Andres and Thummel, 1995
)
using an RNA Transcription Kit (Stratagene, La Jolla, CA). A PCR fragment
synthesized against the DNA region that identified an mRNA missing in
ida
107 animals was prepared by random labeling
(Prime It II Kit, Stratagene) and used to screen a 0-24 hour
ZAPII
embryonic cDNA library (Hurban and
Thummel, 1993
). To ensure accuracy, positive clones were sequenced
from both strands by the Northwestern University Biotech Facility (Chicago,
IL) using gene-specific primers (Integrated DNA Technologies, Coralville, IA).
The gene structure was determined by aligning cDNA with genomic DNA sequenced
from subclones of the chromosomal walk
(Andres and Thummel, 1995
) and
the completed Drosophila genome
(Adams et al., 2000
). Database
searches were performed using BLAST 2.01
(Altschul et al., 1997
) and
sequence alignments were done using CLUSTALW 1.74
(Thompson et al., 1994
).
Genomic DNA was prepared as previously described
(Gloor et al., 1993). PCR
fragments were amplified from the parental (bw; st) and ida
mutant stocks using the following primer pairs. Primers containing coding
information are presented in codon form. Ida 1AS
(5'-CGGCTGATGATTTTGTGTTT-3') and Ida 1S
(5'-CCTGAGCAAGCAGTACCAAGTGTT-3'), Ida 2AS (5'-T ATG TAC AAG
AAC GTA TGC TCC T-3') and Ida 2S
(5'-AGGCACGTTGGAATAAGGAGTC-3'), Ida 3AS (5'-AT TAC TAC AAT
GCT CTT TCT G-3') and Ida 3S (5'-ATCCGTTTTTGCTCGTTC-3'), Ida
4AS (5'-CAG CAC AGC GAC AAT CTC AC-3') and Ida 4S
(5'-GAACACGTCACATCCAAAAC-3'). PCR products were subcloned into the
pGEM-T Easy vector (Promega, Madison, WI) and sequenced by the
Northwestern University Biotech Facility. Mutations were verified using two
independent rounds of amplification and sequencing.
Generation of ida germline clones
ovoD1/FRT stocks specific for chromosome 3L
(Chou et al., 1993) were used
to generate the following stocks by meiotic recombination:
w1118; idaX, P[w+,
FRT]3L-2A/TM3, Sb (where X is the
107, b4 or
d14 allele of ida). Females from these lines were mated to
y w, P[ry+, Flp122]/Y; P[w+,
ovoD1]3L-2X48, P[w+, FRT]3L-2A/TM3, Sb
males. All progeny from this cross were heat shocked for 2 hours at 37°C
on two consecutive days during second-and third-instar development.
Sb+ females from the above cross were collected and mated
to w1118; Df(3L)GN19/TM6B, Tb e Hu males. The
Df(3L)GN19 deficiency removes DNA from 63F3 to 64B2, including the
entire ida gene (Garbe et al.,
1993
; Zhimulev et al.,
1998
). Embryos from these crosses were collected on standard
molasses plates and observed for viability by scoring hatching. A Dichaete
(D) marked chromosome (w; D, P[w+, FRT]3L-2A/TM3,
Sb) was used as a positive control for FLP/FRT mediated mitotic
recombination.
Cytological characterization
ß-galactosidase activity from the esg-lacZ line was detected
in dissected larval tissues using X-gal (5-bromo-4-chloro-3-indolyl
ß-D-galactopyranoside) as a substrate (Sigma, St Louis, MO) as previously
described (Hoshizaki, 1994).
Tissues were mounted in 80% glycerol and observed under brightfield using a
Zeiss Axiophot Microscope (Carl Zeiss, Thornwood, NY). Images were captured
with a Spot Digital Camera (Diagnostic Instruments, St Sterling Heights,
MI).
For DNA staining, tissues were dissected in PBS (3 mM
Na2PO4, 7 mM Na2HPO4, 130 mM NaCl,
pH 7.2) and bathed in Hoechst 33742 (Sigma) diluted 1:1000 in PBS for 5
minutes. Tissues were washed three times for 5 minutes each in PBS, and
mounted in 80% glycerol. Images were collected as described above using
fluorescence microscopy. For orcein cytology, third-instar larval brains were
dissected and squashed as previously described
(Gatti et al., 1974;
Williams et al., 1992
). A
mitotic index (the total number of cells per brain containing condensed
chromosomes over the total number of fields scored) was determined for each
brain following examination under brightfield.
For antibody staining, brains were dissected, fixed and analyzed for marker
proteins as previously described (Williams
and Goldberg, 1994). Tissues were incubated in primary antibody
overnight at 4°C at the following dilutions: 1:1000 for rabbit anti-Bub1
(Basu et al., 1999
), 1:500 for
rabbit anti-centrosomin (Heuer et al.,
1995
), 1:5 for mouse anti-cyclin B
(Knoblich and Lehner, 1993
;
Edgar et al., 1994
), and 1:100
for rabbit anti-
-tubulin (Amersham, Buckinghamshire, UK). Goat
anti-mouse or goat anti-rabbit secondary antibodies conjugated to FITC or
Texas Red (Jackson Immuno Research, West Grove, PA) were diluted (1:250) and
incubated with tissues overnight at 4°C. Samples were washed in PBT
(PBS+0.3% Triton X-100), incubated in Hoechst 33742 (described above), air
dried, and mounted in 80% glycerol in 1 M Tris, pH 8.8 with 1%
n-propyl-gallate. Confocal images were captured with a Zeiss Axiovert 100 M
confocal microscope system (Carl Zeiss).
For RNA interference (RNAi), a full-length ida cDNA was cloned
into pBS-SK(minus) (Stratagene) and amplified using T3 and T7
primers. RNA was transcribed from the PCR product in both directions using the
Ribomax RNA kit (Promega) and annealed to make double stranded RNA (dsRNA) as
previously described (Fire et al.,
1998). Drosophila Kc tissue culture cells
(Echalier and Ohanessian,
1970
) were diluted to 1x106 cells/ml in M3 media
(Hyclone Laboratories, Logan, UT) and incubated with dsRNA as previously
reported (Clemens et al.,
2000
), except that serum was not used. Cells were collected after
48 hours, treated with colchicine (Sigma) and hypotonic solution (0.5% sodium
citrate), and fixed as previously described
(Pimpinelli et al., 2000
).
Cells were dropped onto slides, air-dried and mounted in 80% glycerol
containing 0.5 µg/ml Hoechst 33742 and 2% n-propyl gallate. Images were
captured using fluorescence microscopy as described above.
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Results |
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Though embryonic and larval development is normal
(Vaskova et al., 2000), all
ida mutants reported here (ida
107,
idab4, idad14 and idak1-449)
stall in metamorphic development between 3 and 6 hours after puparium
formation (APF). Fig. 1A,B
compares a wild-type animal at 48 hours APF to an ida mutant of the
same age in which gas bubble migration (an early prepupal event) has stalled
(arrow, Fig. 1B). Although the
point at which ida mutants stall during development is distinct, the
time of lethality, as scored by non-pulsation in the dorsal vessel, ranges
from 24 to 72 hours APF.
|
To determine the cause of lethality, we dissected
ida107, idab4, idad14 and
idak1-449 homozygous wandering third-instar larvae. Data
for the ida
107 deficiency is presented here,
although all homozygous and hemizygous null mutants display identical
phenotypes. Brightfield images of dissected tissue show that ida
mutants lack mature imaginal discs, the clusters of diploid cells that give
rise to the adult structures (compare Fig.
1C and D). In addition, the optic lobes in ida mutant
brains are drastically reduced in size compared with wild-type (arrows,
Fig. 1C,D). This observation is
consistent with the absence of imaginal tissues, as the optic lobes contain
epithelial cells (proliferation zones) that rapidly divide during larval
development (Ito and Hotta,
1992
).
As a more sensitive method for identifying imaginal disc cells, we examined
escargot (esg) expression in ida+ and
ida animals using the esg-lacZ reporter construct.
esg serves as a general marker for diploid imaginal disc cells
(Hayashi et al., 1993).
Dissected tissues from ida homozygotes and heterozygous control
siblings carrying at least one copy of esg-lacZ were examined for
ß-galactosidase (ß-gal) activity in wandering third-instar larval
stage. In control larvae heterozygous for ida, ß-gal activity is
observed in the optic lobes, as well as in the excorporate imaginal discs
(arrowheads, blue staining, Fig.
1C) in a pattern that has been previously reported
(Hayashi et al., 1993
).
Although mature imaginal discs are never detected in ida larvae,
small groups of ß-gal producing cells in close proximity to the brain are
detected (arrowheads, Fig. 1D).
We propose that these esg-lacZ expressing cells are rudimentary
excorporate imaginal discs that have failed to proliferate during larval
stages.
We also examined the developmentally divergent incorporate imaginal discs by examining the imaginal rings. These structures in the gut and salivary gland are easily detected by staining whole tissues with Hoechst to distinguish small diploid imaginal nuclei from larger polyploid larval nuclei. Using this method, we show that a mature imaginal ring in the anterior gut is absent in ida homozygous mutants (compare arrows in Fig. 1E and F). esg-lacZ expression is observed in only 2-3 cells in the anterior gut imaginal ring from ida homozygotes (arrows, Fig. 1H). A similar reduction is seen in imaginal rings of the hindgut, salivary glands, and in the cells of the imaginal midgut islands (data not shown). These data show that mutations in ida cause universal growth defects in both types of imaginal discs during larval development.
To distinguish between a failure of imaginal cells to proliferate versus a
failure to establish the correct primordial cell number, we carefully examined
the fate of histoblasts the nests of cells that form the abdominal
cuticle of the adult. Histoblast cells are established during embryogenesis,
but they initiate proliferation after puparium formation
(Garcia-Bellido and Merriam,
1971). Therefore, the number of abdominal histoblast cells present
at third-instar stage represents the number of cells established during
embryogenesis. Thus, we compared the number of ß-gal staining cells
(using the esg-lacZ reporter) in each histoblast nest of wild-type
and ida mutant animals at the third-instar larval stage. After
examining at least 50 abdominal segments from ida mutants, we
determined that the number of cells in the anterior dorsal, the posterior
dorsal, and the ventral nests is the same as that reported for wildtype
(Merriam, 1978
;
Hama et al., 1990
;
Hartenstein et al., 1992
).
Fig. 1I,J compares
representative examples of the anterior-dorsal (ad) and posterior-dorsal (pd)
nests from wild-type and ida animals. These data suggest the proper
number of progenitor cells is established in ida mutants, but these
cells fail to proliferate.
ida encodes a homolog of APC5
In order to identify the gene product responsible for the ida
phenotype, RNA probes were synthesized against genomic subclones from a 63F
chromosomal walk (Fig. 2A).
These probes were hybridized to northern blots containing samples from
wild-type and ida mutant animals. Strand-specific riboprobes from
p63F.15 hybridize to three mRNA species of 2.3 kb, 1.2 kb, and 1.1 kb
in wild-type animals, but fail to hybridize to the 2.3 kb mRNA species in
ida107 animals
(Fig. 2B). Animals homozygous
for a
30 kb deficiency (Df(3L)449) die as first-instar larvae,
and are also missing the 2.3 kb transcript (data not shown). However, this
early lethality can be rescued to the prepupal period with the introduction of
a 10 kb genomic fragment (P[w+, 63F/k1]) as shown
in Fig. 2A. These animals
(designated idakl-449) display the ida phenotype.
Although they contain a complete ida open reading frame, essential
regulatory information is not present on the transgene, and the expression of
the 2.3-kb mRNA is drastically reduced in idakl-449
animals (Fig. 2B). Although
idakl-449 animals behave as genetic nulls, this molecular
data suggest that they are severe hypomorphs because they might produce some
IDA protein.
|
In order to clone the ida gene, a 0-24 hour embryonic cDNA library
was screened using a probe generated from the interval missing in the
ida107 deficiency
(Fig. 2A). Two overlapping
clones that share a common 3' end including a polyA tail were isolated.
The longer (2.375 kb) clone most likely represents a full length ida
cDNA, as it originates
400 nucleotides from the 3' end of the
dSc2 gene (Vaskova et al.,
2000
).
Comparison of cDNA and genomic sequences confirmed the presence of a 62
base pair intron (Fig. 2A;
Fig. 3A). The ida cDNA
encodes a 777 amino acid protein of approximately 88 kDa. IDA displays 23%
identity and 61% similarity to the human APC5 subunit of the APC/C
(Yu et al., 1998). This
identity is along the entire length of both the IDA and APC5 proteins. Also,
GadFly (http://flybase.bio.indiana.edu:82/annot/) predicts a second intron in
addition to the one that we present here (CG10850). Although we did not
recover a cDNA corresponding to this splice variant in our initial screen, we
note the possibility that a second protein isoform of IDA may exist. However,
this intron would remove coding information for 36 amino acids that has 41%
similarity to the human APC5.
|
A single transcriptional unit with similarity to APC5 is present in the
Drosophila genome, suggesting that IDA is the only APC5 homolog in
flies. Putative APC5 homologs are also found in S. cerevisiae
(YOR249C) and Caenorhabditis elegans (T23780), with identities of 12%
and 14%, respectively. Also consistent with it encoding an APC/C subunit, IDA
contains a tetratricopeptide repeat (TPR) domain at residues 334-367 (blue
box, Fig. 3A). A single TPR
domain is also predicted for APC5 homologs in H. sapiens, S.
cerevisiae and C. elegans
(Fig. 3B). TPR domains are
present in other APC/C subunits, and are required for optimal APC/C complex
formation (Sikorski et al.,
1990; Das et al.,
1998
).
We have also characterized the two EMS-induced genetic null alleles of
ida. In idad14 animals, the 2.3 kb transcript is
not detected by northern blot (data not shown). These data are consistent with
the hypothesis that the idad14 molecular lesion affects
the expression or stability of the ida transcript. By contrast, mRNA
levels from idab4 animals are comparable to those of
wildtype (Fig. 2B). Sequencing
of the idab4 allele shows that a nonsense mutation
converts a glutamine at position 709 to a stop codon (CAGTAG),
truncating the last 68 amino acids of the protein (asterisk,
Fig. 3A). Consequently,
putative protein kinase C (PKC) and cAMP-dependent phosphorylation sites (pink
box, Fig. 3A) at residues
723-725 (SFK) and 725-728 (KKLS) respectively, are removed in this allele. Our
finding that idab4 behaves as a genetic null is consistent
with results from several groups showing that both cAMP-dependent and
PKC-dependent post-translational modifications of APC/C subunits play
essential regulatory roles (Yamashita et
al., 1996
; Yamada et al.,
1997
; Kotani et al.,
1998
). In addition, since idab4 homozygotes
are devoid of imaginal discs, the near wild-type levels of mRNA observed in
this mutant demonstrate that ida expression is not limited to diploid
imaginal tissues.
ida+ is required for oogenesis and/or germline
proliferation
We have demonstrated that zygotic ida+ is not essential
for the establishment of imaginal primordia or developmental events during
embryogenesis. However, it is well known that a maternal contribution of many
gene products can sustain early embryonic development (reviewed by
Foe et al., 1993). A profile
of mRNA expression throughout development shows that ida is highly
expressed during early embryogenesis (data not shown). Based on that
expression data, we suspected that IDA may play a role in cell cycle
progression during embryogenesis, and therefore we investigated whether
embryos lacking the maternal component of ida
(idamaternal) display cell cycle defects during embryonic
development.
Thus, we examined the phenotype of embryos lacking
idamaternal using the ovoD1
female-sterile mutation (Chou et al.,
1993; Chou and Perrimon,
1996
) and FLP-FRT technology
(Golic and Lindquist, 1989
).
For this analysis, ida/ida mutant germlines were produced after
mitotic recombination (see Materials and Methods). All three ida
alleles examined produce identical results. As expected, when wild-type
germline clones (ida+
ovo+/ida+ ovo+)
are produced by mitotic recombination, a cohort of control females produces
several thousand eggs. However, when ida germline clones are produced
(ida ovo+/ida ovo+), a similar cohort
of females produces 3-5 eggs, all of which fail to hatch
(Table 1). Since the three
ida alleles analyzed were generated from separate mutageneses and
parental chromosomes, these results are not likely caused by other mutations
made homozygous in the germline clones. These data suggest that IDA is
essential for mitotic and/or meiotic events during oogenesis and early
embryogenesis. We also note male germline defects, as testes formation is
severely reduced in third-instar male larvae homozygous for ida (data
not shown).
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ida mutant cells have a high mitotic index and overcondensed
chromosomes
We have demonstrated that proliferating cells of the imaginal discs, optic
lobes and germline are affected by mutations in ida. In an effort to
understand how IDA normally functions in the diploid cell cycle, we examined
brains squashes of ida third-instar larvae for specific mitotic
defects.
Initially, a mitotic index measuring the proportion of cells with condensed
chromosomes was determined for each brain scored (see Materials and Methods).
Mutations affecting the cell cycle often affect the mitotic index, as these
cells have difficulty entering and/or exiting mitosis. Brain squashes from
ida third-instar larvae show that the mitotic index is increased more
than threefold when compared with wild-type
(Table 2). The frequency of
prometaphase figures is also increased by at least threefold in mutants. The
magnitude of this increase in mitotic index is consistent with that observed
in several mutants that cause mitotic arrest
(Gatti and Baker, 1989). In
addition, chromosomes in ida cells are highly overcondensed compared
with wild-type (Fig. 4A-D).
This is a typical consequence of mitotic arrest
(Gatti and Baker, 1989
) and
demonstrates that ida mutant cells can enter mitosis successfully,
but that their progression through mitosis is delayed or prevented.
|
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In wild-type squashes, prometaphase can easily be distinguished from metaphase figures in which chromosomes are orientated along the equatorial plate in the center of the cell. In ida brain squashes, prometaphase-like figures are often observed (Fig. 4C). However, during the examination of 2000 fields, metaphase figures in which chromosomes are fully aligned on the central metaphase plate were never detected. This is in contrast with wild-type brain squashes in which we observed aligned metaphase chromosomes in approximately 8% of cells.
Despite the absence of chromosome congression to a metaphase plate, ida mutant cells appear capable of undergoing anaphase. Anaphase stages are defined as those containing separated sister chromatids and/or two separate chromosome populations displaced towards opposite ends of the cell (Fig. 4B,D). We note that lagging chromosomes are often observed in these anaphase-like figures from ida cells (arrow, Fig. 4D). Such results are somewhat surprising because the APC/C is known to regulate entry into anaphase; thus mutations in APC/C subunits are predicted to prohibit anaphase onset.
One potential explanation for the aberrant anaphases and the total absence
of metaphase figures is that ida mutations affect the centrosome
machinery or spindle microtubule integrity. To address these possibilities, we
examined mitotic spindle morphology and centrosome positioning using
antibodies against tubulin and centrosomin. In both wild-type and ida
mutant mitotic cells, centrosomes have duplicated and properly migrated to
opposite ends of the cell as determined by centrosomin localization (green,
Fig. 4E,F) (Heuer et al., 1995). Overall
microtubule spindle integrity is also maintained (white,
Fig. 4E,F). We observe normal
centrosomin staining and microtubule morphology even in ida mutant
cells containing overcondensed and scattered chromosomes
(Fig. 4F).
ida mutants are aneuploid
Wild-type Drosophila mitotic cells have eight chromosomes, while
the vast majority of ida mutant cells contain many more than eight
masses of highly condensed chromatin (Fig.
4C,D). One possible explanation for this observation is that
chromosomes are fragmented and scattered throughout the spindle as acentric
masses. To address whether ida mutations cause chromosome
instability, we prepared orcein brain squashes from
idak1-449 third-instar larvae. In 1% of the mitotic cells,
chromosome hypercondensation in not observed, perhaps because
idak1-449 animals produce low levels of ida mRNA
(Fig. 2B). In these cases, we
observe an excessive number of chromatin figures
(Fig. 5A,B). However, each
chromatin figure is an unfragmented chromosome. In addition, we examined null
mutants that display overcondensed chromosome-like bodies using an antibody
against Bub1 to mark kinetochores. As shown in
Fig. 5C,D, each overcondensed
chromatin figure contains Bub1 signal, and is therefore not an acentric
chromosomal fragment. As the severity of aneuploidy is similar for each of the
four ida alleles that we describe here, we conclude that aneuploidy,
and not general chromosome instability, contributes to the significant
increase in the number of chromatin structures that we observe in ida
mutants.
|
Upstream spindle checkpoint events are active in ida
cells
In an effort to determine whether the spindle checkpoint pathway is active
in ida cells, we examined Bub1 protein localization in mutants as
described above. In wild-type cells, Bub1 protein normally localizes to
kinetochores during prometaphase and metaphase, but staining is reduced in
intensity during anaphase (compare cells marked with asterisks to those marked
with brackets, Fig. 6A,D). Thus, Bub1 staining can serve as a marker for an active spindle checkpoint
pathway and blocked APC/C activity (Taylor
and McKeon, 1997; Skoufias et
al., 2001
). However in ida cells, the intensity of Bub1
staining at the kinetochores is unchanged during all cell cycle stages,
including those with an anaphase-like morphology (asterisks and brackets,
Fig. 6B-C,E-F). An identical
staining pattern is observed with Bub3, another component of the spindle
checkpoint pathway (data not shown). One explanation for the persistence of
Bub1 and Bub3 staining in ida cells is that the stages we classify as
anaphase are actually prometaphase. However, our classification of anaphase is
based on the classic hallmarks of poleward movement of chromosomes, spindle
elongation and sister-chromatid separation, which all occur in ida
mutant cells. An alternative explanation for the persistence of Bub1 and Bub3
in ida cells is that the spindle checkpoint pathway remains active in
these mutants during anaphase events (see Discussion).
|
Sister-chromatid separation occurs in ida mutant cells
Securin proteins are inhibitors of sister-chromatid separation; thus their
destruction is required for anaphase onset and is dependent on the activation
of the APC/C by Fizzy (Hixon and
Gualberto, 2000). Sister-chromatid separation is not expected in
cells deficient for APC/C components; however, the large number of chromatin
bodies in ida mutant cells can be partly explained if each body
represents an individual sister chromatid.
To address whether mutations in ida affect Securin degradation, we
assayed sister-chromatid separation in idak1-449 brain
squashes where the chromosomes are not hypercondensed. In these mutants,
sister chromatids often appear separated with no obvious centromeric
connection (arrows, Fig. 7A).
However, since the hypercondensation of chromosomes in the other ida
mutants makes it extremely difficult to resolve individual chromatids, we
examined karyotypes from Drosophila tissue culture cells treated with
double-stranded RNA (dsRNA). RNA interference (RNAi) by dsRNA treatment is an
effective means to silence genes in Drosophila (reviewed by
Carthew, 2001), and has been
shown to work in cell culture (Clemens et
al., 2000
). As shown in Fig.
7B-C, treatment of Kc cells with dsRNA specific for ida
results in many of the phenotypes we observe in ida mutant brains.
The number of cells displaying discrete, condensed chromosomes is roughly
three times higher in RNAi-treated cells than in controls (data not shown).
Also, in the majority of these chromosome spreads, the chromatin is
overcondensed compared with untreated controls. Chromosomal DNA is in the form
of bi-lobed structures with a constriction in the middle and, in rare cases,
we observe structures with four lobes. Careful examination shows that these
4-lobed structures are chromosomes with connected sister chromatids (arrow,
Fig. 7C), while the more
frequent bi-lobed structures correspond to separated chromatids (arrowheads,
Fig. 7C). These observations
are noteworthy as they suggest that IDA is not essential for sister-chromatid
separation. Therefore, cells mutant for ida are not blocked at
anaphase onset, and potentially may proceed through anaphase and
cytokinesis.
|
Cyclin B degradation does not occur in ida mutant cells
The first step of anaphase onset, sister-chromatid separation, can occur in
cells depleted for IDA function. In an effort to understand the extent to
which ida mutant cells become committed to anaphase events, we
examined protein levels of a second APC/C target molecule, cyclin B. Cyclin B
degradation is a regulated even that is dependent on the Fizzy and
Fizzy-related activation of APC/C (Murray,
1995), and mutations in the CDC16 and CDC27
APC/C subunits result in high B-type cyclin-dependent kinase activity
(Heichman and Roberts, 1996
).
Thus, we wanted to determine whether cells mutant for ida also
contain elevated levels of cyclin B.
In wild-type, cyclin B levels are high during prometaphase and metaphase (Fig., 8A,E) and then diminish during anaphase (Fig. 8C,G). In an ida mutant, cyclin B levels are also high during prometaphase (Fig. 8B,F), but 84% of cells scored as anaphase (with elongated spindles and resolvable groups of chromatids displaced toward the two poles) show levels of cyclin B comparable to levels observed at prometaphase (Fig. 8D,H; Table 3). Thus, cyclin B levels remain high in most ida cells, including cells with segregating chromosomes that appear to be undergoing anaphase.
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Discussion |
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---|
A molecular characterization of ida shows that it encodes a
protein with 23% identity and 61% similarity to APC5 from H. sapiens
(Yu et al., 1998), a subunit
of the APC/C. The APC/C is known to play essential roles in the ubiquitination
of Securins and B-type cyclins. The degradation of these proteins is required
for anaphase progression and exit from mitosis
(Wheatley et al., 1997
;
Page and Hieter, 1999
).
Mutations in some APC/C subunits exist in S. cerevisiae and S.
pombe that cause inviability and defects in cell cycle progression. These
observations are consistent with the ida phenotype that we describe
here. Our data support the theme that yeast and Drosophila share a
functionally conserved set of APC/C proteins that regulate cell cycle
progression and exit from mitosis.
Because ida cells contain condensed chromosomes, they can enter mitosis. However, the high mitotic index seen in brain squashes (Table 2) suggests that cells have problems exiting mitosis. Both prometaphase and anaphase figures are frequently observed in ida mutants (Table 2) even though chromatin figures are severely hypercondensed (Fig. 4C,D). ida cells are aneuploid (Fig. 5), and since the number of chromosomes is rarely a multiple of 8, this aneuploidy is probably a result of missegregated chromosomes during cell division. Thus at some level, ida cells are capable of progressing through the cell cycle.
Only a subset of APC/C-dependent events is compromised in ida
mutants. For example, in ida mutant cells attempting anaphase, cyclin
B levels remain high (Fig.
8D,H; Table 3).
Consistent with these data, the hypercondensed chromosome figures in
ida mutants are strikingly similar to those observed when a
non-degradable form of cyclin B is overexpressed in proliferating cells
(Rimmington et al., 1994). By
contrast, other known APC/C-dependent events such as sister-chromatid
separation can occur upon IDA depletion
(Fig. 7A,C). This demonstrates
that mutations in ida do not effect the APC/C-dependent degradation
of Securin proteins. The fact that IDA is required for a fraction, but not all
of the APC/C functions suggests that it does not play an essential role in the
stability of the core complex, or in the ligation of ubiquitin oligomers to
substrates.
Subfunctions of the APC/C
In Drosophila the APC/C complex is estimated to consist of 11
proteins. However the biochemical function and requirement of so many subunits
is unclear. One hypothesis proposes that the large number of subunits reflects
the need to identify and target a large number of substrates. The model is
supported by the recent characterization of the 3D structure of the human
APC/C (Gieffers et al., 2001).
The structure has an asymmetric morphology with a large inner cavity
surrounded by an outer protein wall. The complexity of the structure suggests
that discrete subunits may guide substrates into the inner cavity, where
ubiquitination could take place. Thus the removal of a single subunit would
disrupt the ubiquitination of only a fraction of substrates. Interestingly,
our data suggests that IDA may be involved in the degradation of cyclin B but
is not essential for the degradation of Securins. It should be noted that in
this model not all subunits need play a role in substrate identification, as
some are required for core stability and catalyzing the ubiquitination events.
For example, Cdc27 and Cdc16 play critical roles in core stability
(King et al., 1995
), and Apc11
is required for the ubiquitination of substrates
(Gmachl et al., 2000
;
Ohta et al., 1999
).
Another consideration for the role of APC/C subunits concerns the
possibility that they specifically interact with regulators of APC/C activity
during the cell cycle. Perhaps the most actively studied regulators of APC/C
activity are the components of the spindle checkpoint pathway. Upon detection
of DNA damage or unattached kinetochores, the spindle checkpoint pathway will
send a `wait' signal. In response to this signal, Mad2 will bind the APC/C,
preventing its activity and halt progression of all mitotic events until the
checkpoint has been fulfilled (Li et al.,
1997; Fang et al.,
1998
). Positive regulators play an equally important role in
driving the cell through coordinated mitotic events. In Drosophila,
the WD40-repeat protein, Fizzy (FZY), binds to and drives APC/C-dependent
ubiquitin-ligase activity in vitro (Kramer
et al., 1998
). The FZY homolog in yeast, Cdc20p, positively
regulates the destruction of Pds1p
(Shirayama et al., 1998
), and
FZY is thought to serve a comparable role in Drosophila because FZY
is required for Pimples (Securin) degradation during mitosis
(Leismann et al., 2000
).
Consistent with these predictions, loss-of-function mutations in fzy
prohibit cells from progressing through metaphase
(Dawson et al., 1993
), and
demonstrate that FZY is required for metaphase exit and completion of mitosis
in Drosophila. FZY is highly unstable and present only at late S
phase and during mitosis, further ensuring that FZY-dependent APC/C events are
temporally regulated. Finally, FZY degradation is dependent on APC/C subunits
(Prinz et al., 1998
),
demonstrating that FZY is also a substrate of the APC/C. An additional
WD40-repeat protein, Fizzy-related (FZR), is also believed to be required for
the degradation of B-type cyclins during M and G1 phases
(Sigrist and Lehner, 1997
),
but differs from FZY in that it is stable throughout the cell cycle
(Prinz et al., 1998
).
Possible roles for IDA in the APC/C
We have shown that in ida mutants, cyclin B levels are not
properly degraded during anaphase. Thus it is possible that the function of
IDA, alone or in concert with other subunits, is to direct cyclin B to the
APC/C for degradation. However, we submit other defects observed in
ida cells that are distinct from those observed in cells expressing a
non-degradable cyclin B transgene
(Rimmington et al., 1994;
Parry and O'Farrell, 2001
).
Therefore, we propose additional regulatory functions for the IDA protein to
help explain the lack of metaphase figures, the observed sister-chromatid
separation, the high levels of Bub1 staining during anaphase, and the
resulting aneuploidy that is observed in ida mutant cells.
In one model, IDA functions as a part of the APC/C that receives a spindle checkpoint `wait' signal. Thus when IDA is missing, the spindle checkpoint signal is not received, but the cell initiates sister-chromatid separation and anaphase onset prematurely. Presumably, this could occur even in the absence of proper chromosome attachment and alignment at the metaphase plate. Thus, metaphase figures would not be observed in ida mutants, but aberrant anaphases containing lagging chromosomes with high Bub1 staining (equal to signal checkpoint firing) would be detected. The missegregation of the unattached chromatids would also lead to cells containing an aneuploid number of chromosomes.
In an alternative model, IDA plays a role in targeting FZY for ubiquitin-dependent degradation. In this case, the removal of IDA would result in ectopic levels of FZY, which would prematurely activate sister-chromatid separation and progression through mitosis. Consistent with this model, mutations in ida suppress the embryonic lethality associated with a fizzy null mutation (A.M.B. and A.J.A., unpublished).
It should be noted that neither model directly address the high mitotic index a hallmark of cell cycle stall observed in squashes of ida cells. However, we propose that as cells become more and more aneuploid, alternative pathways, including the DNA replication checkpoint, may eventually cause a prometaphase stall or arrest.
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References |
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