Download Structurally related TPR subunits contribute differently to the function

3238
Research Article
Structurally related TPR subunits contribute
differently to the function of the anaphase-promoting
complex in Drosophila melanogaster
Margit Pál, Olga Nagy, Dalma Ménesi, Andor Udvardy and Péter Deák*
Institute of Biochemistry, Biological Research Center, H-6726 Szeged, Hungary
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 10 July 2007
Journal of Cell Science 120, 3238-3248 Published by The Company of Biologists 2007
doi:10.1242/jcs.004762
Summary
The anaphase-promoting complex/cyclosome or APC/C is
a key regulator of chromosome segregation and mitotic exit
in eukaryotes. It contains at least 11 subunits, most of
which are evolutionarily conserved. The most abundant
constituents of the vertebrate APC/C are the four
structurally related tetratrico-peptide repeat (TPR)
subunits, the functions of which are not yet precisely
understood. Orthologues of three of the TPR subunits have
been identified in Drosophila. We have shown previously
that one of the TPR subunits of the Drosophila APC/C,
Apc3 (also known as Cdc27 or Mákos), is essential for
development, and perturbation of its function results in
mitotic cyclin accumulation and metaphase-like arrest. In
this study we demonstrate that the Drosophila APC/C
associates with a new TPR protein, a genuine orthologue of
the vertebrate Apc7 subunit that is not found in yeasts. In
addition to this, transgenic flies knocked down for three of
the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by
RNA interference were established to investigate their
function. Whole-body expression of subunit-specific
dsRNA efficiently silences these genes resulting in only
residual mRNA concentrations. Apc6/Cdc16 and
Apc8/Cdc23 silencing induces developmental delay and
Key words: APC/C, TPR subunits, Apc6 (Cdc16), Apc7, Apc8
(Cdc23), Transgenic RNAi
Introduction
The ubiquitin-dependent protein degradation system controls
mitotic progression by sequential degradation of different
regulatory proteins. In this process a pathway of ubiquitin
activating (E1), ubiquitin conjugating (E2) and ubiquitin ligase
(E3) enzymes catalyse the conjugation of polyubiquitin chains
onto substrate proteins, leading to their recognition and
degradation by the 26S proteasome. The key component of this
proteolytic system is a multi-subunit ubiquitin ligase, the
anaphase-promoting complex (APC/C) that provides a
platform and specificity for the ubiquitination reactions. The
APC/C is active during mitosis and G1 phases of the cell cycle
in which, by mediating securin and mitotic cyclin degradation,
it triggers the metaphase to anaphase transition and the exit
from mitosis (Harper et al., 2002).
The APC/C has been purified and analysed biochemically
from yeast, Xenopus and clam egg extracts and from human
cells. It turned out to be a large protein complex containing at
least 13 stably associated core subunits in yeasts (Passmore,
2004). Orthologues of ten of the yeast subunits could be
identified in higher eukaryotes as well, indicating that the
structure of APC/C is evolutionarily conserved. Only the Apc9
subunit appears to be specific to unicellular yeasts, whereas the
Apc7 subunit has so far been identified only in vertebrates
(Harper et al., 2002).
Despite the essential role of APC/C in regulating mitosis, little
is known about the function of most of its individual subunits.
The presence of structurally related proteins to the cullin
homologue Apc2, the RING finger Apc11 and the DOC1
domain containing Apc10 subunits in other E3 ubiquitin ligases
suggests direct roles of these subunits in the ubiquitination
reaction. Indeed, it was shown that the Apc2 and Apc11 subunits
interact with each other (Ohta et al., 1999) and depending on the
E2 enzyme, Apc11 alone or together with Apc2 could support
the nonspecific transfer of ubiquitin to protein substrates
(Gmachl et al., 2000; Leverson et al., 2000; Tang et al., 2001)
in vitro. Furthermore, Apc10 was suggested as the processivity
factor for the APC/C (Carroll and Morgan, 2002).
Much less is known about the function of other APC/C
subunits, such as Apc1, Apc4 and Apc5. The ida gene coding
causes different pupal lethality. Cytological examination
showed that these animals had an elevated level of
apoptosis, high mitotic index and delayed or blocked
mitosis in a prometaphase-metaphase-like state with
overcondensed chromosomes. The arrested neuroblasts
contained elevated levels of cyclin B but, surprisingly,
cyclin A appeared to be degraded normally. Contrary to
the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the
apparent loss of Apc7 function does not lead to the above
abnormalities. Instead, the Apc7 knocked down animals
and null mutants are viable and fertile, although they
display mild chromosome segregation defects and
anaphase delay. Nevertheless, the Apc7 subunit shows
synergistic genetic interaction with Apc8/Cdc23 that,
together with the phenotypic data, assumes a limited
functional role for Apc7. Taken together, these data suggest
that the structurally related TPR subunits contribute
differently to the function of the anaphase-promoting
complex.
Journal of Cell Science
TPR subunits of the APC/C have different functions
for the Drosophila homologue of Apc5 has been cloned and
characterised. Mutant alleles of ida show a characteristic
mitotic phenotype suggesting that this subunit controls some
sub-functions of the APC/C (Bentley et al., 2002).
The Apc3 (also known as Cdc27 or Mks; hereafter referred
to as Apc3/Cdc27/mks), Apc6 (also known as Cdc16; referred
to as Apc6/Cdc16), and the Apc8 (also known as Cdc23; referred
to as Apc8/Cdc23) subunits constitute a group of structurally
related proteins within the APC/C, all of which contain nine to
ten copies of the tetratrico-peptide repeat (TPR) motifs in
tandem arrays. The TPRs are repeats of 34 amino acid structural
motifs with a consensus sequence restricted only to eight
residues. There is no invariant residue even within the consensus
but amino acids at these positions are conserved in terms of size,
hydrophobicity and spacing (Lamb et al., 1995; Blatch and
Lässle, 1999). The first X-ray structure of the TPR containing
protein phosphatase-5 revealed that each motif forms two ␣helices in an antiparallel, helix-turn-helix configuration (Das et
al., 1998). The neighbouring motifs are packed in a parallel
fashion resulting in the formation of a superhelical structure.
TPR motifs are present in functionally divergent proteins and
thought to mediate protein-protein interactions and the assembly
of multiprotein complexes. It is believed that the TPR subunits
of the APC/C form a scaffold-like structure that facilitates the
binding of other catalytic subunits, regulatory proteins and
substrates (Passmore et al., 2005). TPR subunits are essential for
viability in yeasts, and their mutant alleles result in uniform,
mitotic G2-M arrest phenotype (Lamb et al., 1994).
In Drosophila, the genes coding for three TPR subunits,
Apc3/Cdc27/mks, Apc6/Cdc16 and Apc8/Cdc23 have previously
been identified (Deak et al., 2003; Harper et al., 2002; Huang
and Raff, 2002). Characterisation of Apc3/Cdc27/mks mutants
indicated that this subunit is essential for APC/C function and
required for the degradation of both cyclin A and cyclin B (Deak
et al., 2003). In another study, GFP-tagged Apc3/Cdc27/Mks
and Apc6/Cdc16 subunits localised differentially in living
syncytial embryos. Individual depletion of these subunits in
cultured Drosophila S2 cells by RNA interference resulted in
morphologically distinct mitotic phenotypes. These findings
suggest the existence of multiple forms of the APC/C with
different functions (Huang and Raff, 2002).
To better understand the role of the TPR subunits in
maintaining the structure and function of APC/C, we have
initiated a reverse genetic analysis on their homologues in
Drosophila melanogaster. First, the putative Drosophila
orthologue of the human gene Apc7 was identified through
extensive sequence analysis, and then found to associate with
the APC/C complex. Following that, transgenic lines were
constructed carrying inducible RNA interference (RNAi)
constructs specific for the Apc6/Cdc16, Apc7 and Apc8/Cdc23
genes. We show that knocking down these genes results in
strikingly different phenotypes that cover viability, cell cycle
progression, substrate degradation and apoptosis. These
findings suggest that the TPR subunits contribute differentially
to different sub-functions of the APC/C.
Results
The CG14444 gene encodes the Drosophila homologue
of Apc7
The Drosophila genome project (BDGP) identified a gene
designated as CG14444 and localized to the 6C1 region of the
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X chromosome. EST sequences (LD05103 and LD39177)
corresponding to this gene encode a 595 amino acid TPRcontaining protein of approximately 68 kDa. Extensive
sequence comparisons, using BLAST, gapped BLAST and
PSI-BLAST programs (Altschul et al., 1997), showed 30%
identity and 51% similarity of the predicted protein along its
entire length to the Apc7 subunit of the human APC/C (Yu et
al., 1998). Interestingly, predicted proteins from Arabidopsis
(accession no. NP 850309) and Apis mellifera (accession no.
XP 396165) came up with very similar topology to both the
human and Drosophila proteins. In each of these proteins, the
number, location and the distribution of the TPR motifs were
very similar: they all contained eight TPR motifs in a tandem
array at their C terminus and two single repeats toward their
N-terminal end (Fig. 1A). Comparison of the ten TPR motifs
of the CG14444 gene product to each other showed similarity
mostly confined to the consensus sequence of this motif (Fig.
1B). However, sequence comparison of individual motifs to
positionally equivalent TPR motifs of related proteins from
other species revealed that the conservation among these
repeats extends outside of the consensus (Fig. 1C), suggesting
functional correspondence. The similarity pattern outside the
consensus is specific to individual TPRs. Thus these
observations suggest that the CG14444 gene encodes a putative
Apc7 protein in Drosophila melanogaster. Likewise, Apc7
orthologues appear to exist in Apis mellifera and Arabidopsis
thaliana as well, suggesting that this protein is present in all
multicellular eukaryotes. Owing to the high degree of
structural and sequential correspondence between CG14444
and the genuine vertebrate Apc7, we designate the Drosophila
gene as Apc7 in the rest of this paper.
Apc6/Cdc16- and Apc8/Cdc23-specific dsRNA
expression causes larval-pupal polyphasic lethality
The inducible transgenic RNAi approach summarized in Fig.
2A was applied to knock down the expression of the
Apc6/Cdc16, Apc7 and Apc8/Cdc23 genes. Constructs were
made in the pWIZ vector (Lee and Carthew, 2003) in which
inverted doublets of cDNA sequences were placed under the
control of the GAL4-UAS system, and transgenic lines were
established after transformation of isogenic flies. Expression
from these constructs leads to the synthesis of RNAs that are
predicted to snap back after splicing, and form loopless
dsRNAs. Several independent transgenic lines carrying single
copy insertions were isolated for each construct and mapped
to the major chromosomes. All homozygous viable lines
showed comparable results.
To induce expression of transgenes, the RNAi strains were
crossed to strains carrying the Act5C-GAL4 driver with a
ubiquitous expression of GAL4 throughout development (The
FlyBase Consortium, 2003). RT-PCR was used to determine
the expression of endogenous TPR subunit-specific mRNAs.
As shown on Fig. 2B, induction of all three dsRNA syntheses
was able to reduce TPR-specific mRNA levels by ~90% in
early pupae relative to uninduced controls when normalized to
the rpL17A calibration control. The da-GAL4 driver was also
used for RNAi induction, and provided similar silencing effects
to Act5C-GAL4 (data not shown).
To examine how silencing of the TPR subunits affects
viability, defined numbers of first instar larvae carrying single
copies of Act5C-GAL4 and either UAS-Apc6/Cdc16RNAi or
Journal of Cell Science
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Journal of Cell Science 120 (18)
Fig. 1. Distribution and sequence comparisons of TPR motifs in
putative Apc7 orthologues. (A) Distribution of TPRs in the human
Apc7 subunit (Hs), the Drosophila CG14444 gene product (Dm), and
predicted proteins from Arabidopsis thaliana (At; accession no. NP
850309) and Apis mellifera (Am; accession no. XP 396165). The
asymmetric rectangular symbols represent single TPR motifs.
(B) Sequence alignment of seven TPR motifs of the Drosophila
Apc7 protein. The TPR consensus sequence is shown below the
alignment. Residues matching the consensus are in bold type and
highlighted in grey (also in C). Residues are conserved only at the
consensus positions but, even there, there is no invariant position.
(C) Sequence comparison of the TPR10 motif from the proteins
shown at the top of this figure. Conserved residues outside the
consensus are highlighted in yellow. Residue conservation is more
extensive both within and outside of the consensus.
UAS-Apc8/Cdc23RNAi constructs were collected and their
survival was monitored by counting the number of third instar
larvae and pupae. Induction of Apc6/Cdc16- and Apc8/Cdc23specific RNAi did not significantly affect survival of embryos
and early larvae. However, lethality was observed in the last
larval (L3) stage, and all animals died as pupae. In addition to
this, the third instar larvae showed a 1- to 2-day delay in
puparium formation and frequently these larvae formed
melanotic tumours (Table 1A). No melanotic tumours or
premature deaths were observed in control animals carrying
only a single copy of Act5C-GAL4. The late lethal phenotype
is a characteristic feature of mitotic regulatory genes (Gatti and
Baker, 1989) and shows that the Apc6/Cdc16 and Apc8/Cdc23
subunits are essential components of the Drosophila APC/C.
Although the extent of silencing judged by RT-PCR was
comparable for both Apc6/Cdc16 and Apc8/Cdc23, a clear
distinction could be made in their lethal phenotypes. Whereas
pupae from five independent Apc8/Cdc23RNAi lines died at the
end of the prepupal metamorphosis in the P4(ii) (moving
bubble) stage, animals from all independent Apc6/Cdc16RNAi
lines developed further and died during the first part of the
phanerocephalic pupal (malpighian tubules migrating) stage of
P5(i) (Bainbridge and Bownes, 1981). The Apc8/Cdc23specific RNAi effect was also more pronounced both in terms
of larval lethality and formation of melanotic tumours.
However, both Apc6/Cdc16RNAi and Apc8/Cdc23RNAi animals
died earlier than the ones homozygous for the mks1 allele of
the Apc3/Cdc27/mks gene. The mks1 mutation causes
comparable reduction (~90%) of gene expression compared
with the RNAi knock down effect in Apc6/Cdc16RNAi and
Apc8/Cdc23RNAi animals, yet these mutants die as late pharate
adults with severe rough eye and bristle phenotypes (Deak et
al., 2003).
Apc6/Cdc16- and Apc8/Cdc23-specific gene silencing
results in metaphase-like arrest with overcondensed
chromosomes
To determine whether Apc6/Cdc16 or Apc8/Cdc23 silencing
affects cell cycle progression, we examined orcein-stained
brain squash preparations of RNAi induced third instar larvae
for mitotic defects. In many mitotic cells, the chromosomes
showed up as dot-like structures instead of the characteristic
rod-like wild-type chromosomes, and frequently they appeared
either scattered all over the cells or congregated at the
metaphase plate (Fig. 3E-G,I-K). The chromosome
overcondensation indicated that the cells were delayed or
arrested in mitosis, as chromosomes continue condensation
during this time. Accordingly, the proportion of cells in mitosis
was significantly higher, and this leads to about double the
mitotic index (MI) of the wild type (Table 2). Most of the
mitotic cells were in a prometaphase-metaphase-like state, and
at the same time, the number of cells in ana- and telophases
Fig. 2. Gene silencing by RNA interference. (A) Scheme for transgenic RNA interference used in this work to knock down the expression of
the TPR subunits of Drosophila APC/C. Inverted exon repeats of these genes (white bars) were cloned into pWIZ transformation vector under
the control of the UAS promoter (white circles) and transgenic lines were made. These were crossed to the Act5C-GAL4 driver lines that
constitutively expressed the GAL4 transcription activator in all cells throughout development. Progeny that contained both Act5C-GAL4 and
UAS transgenic constructs expressed double-stranded hairpin RNAs that triggered gene silencing. (B) Monitoring TPR transcript levels in
RNAi induced larvae (I) relative to uninduced controls (C). The rpL17A transcript was used as a calibration control. TPR gene-specific
transcript levels appeared significantly lower in all RNAi induced samples.
TPR subunits of the APC/C have different functions
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Table 1. Lethal phase of TPR genes knocked down by RNA interference
Genotype
A
Apc6RNAi
Apc7RNAi
Apc71
Apc8RNAi
B
Apc6RNA-Apc7RNAi
Apc6RNAi-Apc8RNAi
Apc7RNAi-Apc8RNAi
L3 Larvae* (%)
Prepupal stage P3 (%)
Prepupal stage P4(i) (%)
Prepupal stage P4(ii) (%)
Pupal stage P5(i) (%)
6 + mt
Viable
Viable
13 + mt
3
Viable
Viable
20
–
Viable
Viable
–
11
Viable
Viable
65
86
Viable
Viable
15
7 + mt
27 + MT
20 + MT
3
26
28
–
70
70
15
4
2
82
–
–
Journal of Cell Science
*Percentage of all larvae examined. All other values represent percentages of animals reaching at least prepupal stage. 7-10 samples of 100 larvae were
collected and analyzed for each genotype. MT and mt, frequent and sporadic occurrence of melanotic tissue in L3 larvae, respectively.
stayed relatively low. This is reflected in the two- and threetimes higher metaphase-anaphase ratios (M:A) in the
Apc6/Cdc16 and Apc8/Cdc23 RNAi preparations, respectively
(Table 2), and suggests that loss of function of these subunits
leads to metaphase-like delay or arrest.
The scattered distribution of chromosomes in RNAi induced
Apc6/Cdc16 and Apc8/Cdc23 cells were very similar to the
phenotype caused by the Apc3/Cdc27/mks, Apc5/ida and
Apc2/mr mutations (Deak et al., 2003; Bentley et al., 2002;
Reed and Orr-Weaver, 1997).
However, in about 10% of Apc6/Cdc16 and Apc8/Cdc23
RNAi induced mitotic cells the chromosomes appeared to
congregate precisely to the metaphase plate (Table 2 and Fig.
3F,J). This true metaphase stage was quite rare or indeed could
not be detected in Apc3/Cdc27/mks, Apc5/ida and Apc2/mr
mutants, and was significantly higher than that seen in the wild
type (2-3%). The chromosomes at the metaphase plate were
also overcondensed, indicating that the delay or arrest was
maintained during that stage as well.
In some of the arrested cells the chromosomes appeared to
undergo decondensation before attempting to complete
anaphase and cytokinesis (Fig. 3G). It has been suggested that
Fig. 3. Silencing of the Apc6/Cdc16, Apc7 and Apc8/Cdc23 genes by RNAi results in mitotic abnormalities. Wild-type mitotic cells in
prometaphase (A), metaphase (B) and anaphase (C). Neuroblast cells from both Apc6/Cdc16 (E-G) and Apc8/Cdc23 (I-L) knocked down larvae
show metaphase-like arrest with overcondensed chromosomes. The chromosomes in most of the arrested cells appear scattered (E,G,I,K), in
about 10% of mitotic cells the chromosomes are locked at the metaphase plate (F,J). Some degree of chromosome overcondensation could also
be observed in anaphase figures (L) together with irregular chromosome segregation. Some cells appear polyploid (G,K), and they invariantly
show chromosome overcondensation. Overcondensed chromosomes sometimes appear to be in the process of decondensation (G). Induction of
Apc7-specific RNAi causes a mild mitotic phenotype. Neuroblast cells from Apc7RNAi larvae show signs of uneven chromosome condensation
and telomeric fusions (D) and occasionally chromosome bridges in anaphase (H).
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Journal of Cell Science 120 (18)
Table 2. Mitotic phenotypes of TPR genes knocked down by RNA interference
Genotype
A
Canton S
Apc6RNAi
Apc7RNAi
Apc8RNAi
B
Apc6RNAi-Apc7RNAi
Apc6RNAi-Apc8RNAi
Apc7RNAi-Apc8RNAi
Preparations/fields*
Mitotic index (MI)†
Metaphase:anaphase ratio
Chromosomes at metaphase plate (%)‡
Polyploid (%)‡
4/60
4/60
6/70
4/50
1.7±0.3
3.2±0.3
2.1±0.3
3.5±0.4
1.9±0.3
4.2±0.5
1.4±0.4
5.2±0.3
2.7
11.0
2.2
9.7
0
4.6±1.8
0
8.1±0.9
5/90
5/50
4/40
3.5±0.3
4.4±0.4
4.2±0.4
5.5±0.5
6.6±0.8
6.3±0.5
10.0
11.6
9.3
3.0±0.8
7.7±3.0
6.2±2.1
Journal of Cell Science
*The number of preparations examined and microscopic fields screened for mitotic cells. On average, there were about 100-200 cells per field. †Mitotic index
is given as the average number of mitotic cells in an optical field±s.d. ‡This is percentage of mitotic cells ± s.d.
arrested cells could revert to interphase by decondensing their
chromosomes (Gatti and Baker, 1989). Following DNA
replication, such cells become tetraploid, or with the recurrence
of events, polyploid. A proportion of Apc6/Cdc16 and
Apc8/Cdc23 RNAi cells were polyploid, most frequently with
highly overcondensed tetraploid or less frequently octaploid or
higher ploidy chromosome complements (Table 2 and Fig.
3G,K).
In addition to metaphase, irregular chromosome behaviour
could be detected in anaphase as well, which included lagging
chromosomes and chromosome bridges (Fig. 3L).
Apc6/Cdc16 and Apc8/Cdc23 RNAi lines arrest with
high levels of cyclin B but with apparent destruction of
cyclin A
One of the main functions of APC/C is to aid the degradation
of mitotic cyclins. Previously, we have shown that mutations
(mks1 and mksL7123) in the Apc3/Cdc27/mks gene stabilized
both cyclin A and B in the mitotically arrested cells (Deak et
al., 2003) (M.P. and P.D., unpublished). We were interested to
follow mitotic cyclin levels in RNAi induced neuroblasts by
immunostaining with polyclonal antibodies raised against
cyclin A and cyclin B. In wild-type cells, cyclin A could be
detected in prophase and early prometaphase (Fig. 4A-D), but
it became undetectable around metaphase and early anaphase
(Fig. 4E-H). Cyclin B degradation follows that of cyclin A, so
its level declines rapidly in metaphase, and at and after
chromosome segregation (Fig. 5A-H). As expected, both cyclin
A- and cyclin B-specific antisera gave strong staining in the
RNAi induced Apc6/Cdc16 and Apc8/Cdc23 preparations.
Closer inspection revealed that the cells strongly stained by
cyclin A-specific antibody gave rather weak and somewhat
diffuse DNA signal, indicative of being in prophase or early
prometaphase. The majority of cells (80%) arrested in late
prometaphase or in metaphase were not stained by anti-cyclin
A antibody above background level (Fig. 4I-L,M-P). However,
more than 80% of cells that stained intensely with cyclin Bspecific antibody also showed bright staining of DNA at the
metaphase plate and a spindle indicative of being in metaphase
arrest with overcondensed or polyploid sets of chromosomes
(Fig. 5I-L,M-P). Thus, it appears that in terms of cyclin A
degradation, the Apc6/Cdc16 and Apc8/Cdc23 mitotic
phenotypes differ from that of Apc3/Cdc27/mks mutants,
where both cyclin A and cyclin B accumulate (Deak et al.,
2003). The apparent degradation of cyclin A and the
accumulation of cyclin B in cells with diminished Apc6/Cdc16
and Apc8/Cdc23 functions indicate that these subunits may
have lesser roles in cyclin A removal but are required for cyclin
B degradation.
Apc7 is a nonessential protein that associates with the
Drosophila APC/C
As Fig. 2B demonstrates, RNA interference caused significant
reduction in the expression of the Apc7 gene. This effect was
confirmed independently by using quantitative fluorescent
real-time PCR that showed greatly delayed amplification in
Apc7RNAi samples compared with controls (data not shown).
The delayed amplification is consistent with lower
concentration of Apc7 transcripts in those samples. Although
the degree of gene silencing was at least as effective as in the
Apc6/Cdc16 and Apc8/Cdc23 RNAi lines, the Apc7RNAi
animals developed without any delay or abnormalities and
hatched as fertile adults (Table 2).
Orcein-stained brain squash preparations from Apc7RNAi
third instar larvae lack any sign of prometaphase or metaphaselike arrest characteristic of the other TPR subunit mutants.
Instead, chromosomes appeared somewhat more tangled and
exhibited signs of uneven condensation together with frequent
chromosome bridges in anaphase (Fig. 3D,H). The number of
cells in mitosis remained low but the proportion of the
anaphase and telophase figures approximated the number of
cells in prometaphase and metaphase, resulting in a mitotic
index that was similar to that of wild type, and a metaphaseanaphase ratio (M:A) that was lower than in wild type (Table
2). The lower M:A ratio may indicate some delay during
anaphase in the Apc7RNAi cells.
In order to further support the nonessential nature of the
Apc7 gene, a null mutant allele (Apc71) was isolated by
imprecise excision of the P{EPgy2}EY11333 element
localized 63 bp distally from the 3⬘ end of the gene. The
remobilization generated a 1576 bp deletion extending from
the insertion site toward the 5⬘ end of Apc7. This internal
deletion removed about two-thirds of the Apc7 gene, including
the functionally important eight tandem TPR repeats
(Vodermaier et al., 2003). The Apc71 homozygotes appeared
viable and fertile, and could be maintained as a homozygous
stock. Their mitotic phenotype was indistinguishable from that
of Apc7RNAi lines. To determine the role of Apc7 in cyclin
degradation, we monitored cyclin levels in Apc7RNAi and Apc71
neuroblast preparations by immunostaining. We could not
detect abnormal changes in the level and localisation of either
cyclin A or cyclin B in mitotic cells and their turnover appeared
similar to that in wild type (data not shown).
To see whether Apc7 is a genuine component of the
TPR subunits of the APC/C have different functions
of total protein extracts (lanes 1 and 2). These data show that
at least a fraction of Apc7 is clearly associated with the APC/C
in Drosophila, but perhaps not as a core subunit. Possible
interpretations of the low-yield co-purification of Apc7 in the
FLAG-Apc8 pull-down complex is that Apc7 either interacts
only with certain forms of the APC/C complex or it interacts
only transiently with the APC/C.
Apc8/Cdc23RNAi show synergism with Apc6/Cdc16RNAi
and Apc7RNAi
TPR subunits are thought to be involved in protein-protein
interactions to form large protein complexes (Blatch and
Lässle, 1999), and were suggested to interact with each other
within the budding yeast APC/C (Lamb et al., 1994). Since
genetic interaction tests of double mutants are powerful
techniques to investigate functional relationships either
between genes or their protein products, we applied this to our
RNAi lines. Transgenic strains carrying two RNAi constructs
in different combinations were generated and crossed to
Act5C-GAL4 partners to induce gene silencing. Again, defined
number of first instar larvae carrying single copies of Act5CGAL4 and pair-wise combinations of the UAS-
Journal of Cell Science
Drosophila APC/C, we examined physical interaction
between Apc7 and universal APC/C subunits Apc8/Cdc23 or
Apc3/Mks by co-transfection of S2 cells and affinity
chromatography. FLAG-tagged Apc7 (F7) was co-expressed
in S2 cells with either haemagglutinin (HA) epitope-tagged
Apc8/Cdc23 (H8) or Apc3/Mks (H3), and then FLAG-Apc7
and its associated proteins were pulled down from cell extracts
with an anti-FLAG affinity column. Following repeated
washing, bound proteins were eluted with an excess of free
FLAG peptide, and detected by western analysis using
monoclonal anti-FLAG or anti-HA antibodies. Fig. 6 shows
that both HA-Apc3/Mks (lanes 7 and 8) and HA-Apc8 (lanes
3 and 4) were retained on the column with FLAG-Apc7 under
washing condition of 0.15 M NaCl (in 20 mM Tris), but there
was no detectable retention without Apc7 (Fig. 6, lanes 9 and
10). However, in a reciprocal experiment, when FLAG-Apc8
was co-expressed with HA-Apc7, and FLAG-Apc8-associated
proteins were pulled down, only a faint band of HA-Apc7
could be detected (Fig. 6, lane 6), indicating a weak
association of HA-Apc7 with FLAG-Apc8. The expression
levels of the different N-terminal fusion constructs were
comparable in these experiments as judged from western blots
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Fig. 4. Cyclin A is degraded in
RNAi silenced Apc6/Cdc16 and
Apc8/Cdc23 cells. Columns of
images show tubulin (green and
second column), DNA (blue and
third column) and cyclin A (red
and fourth column) localisations
in mitotic cells. Cyclin A staining
is visible in wild-type prophase or
prometaphase (A-D) but it is
undetectable in most of the wildtype metaphase (E-H) cells
(n=12). Similarly to this, in the
majority (80%) of both
Apc6/Cdc16 (I-L) and
Apc8/Cdc23 (M-P) RNAi induced
cells (n=19 and 26, respectively),
there is no detectable cyclin A at
the onset of metaphase, indicating
that cyclin A degradation is not
affected in these lines.
3244
Journal of Cell Science 120 (18)
pronounced mitotic arrests phenotype of the double RNAi lines
suggest a firm synergistic interaction between Apc8/Cdc23 and
Apc6/Cdc16 as well as Apc8/Cdc23 and Apc7, thus implicating
that these subunits contribute independently to the same
function of the APC/C.
The phenotype of the third double RNAi line,
Apc6/Cdc16RNAi-Apc7RNAi appeared different from the ones
discussed above. The lethal phase and mitotic phenotype were
similar to those of Apc6/Cdc16RNAi alone (Table 1), the
hallmark of epistatic interaction. However, as one of the lines,
Apc7RNAi, is viable with mild mitotic phenotype, one would
also expect the double mutant phenotype to reflect the stronger
mutant if these gene products act independently, therefore no
obvious interaction can be established between Apc6/Cdc16
and Apc7.
Apc6/Cdc16- and Apc8/Cdc23-specific RNA interference
induces apoptosis
In addition to mitotic cells, we observed many pycnotic nuclei
in the orcein-stained preparations (Fig. 7A,B). The cells were
rounded, relatively small and their nuclei showed strong but
uneven staining. Pycnotic nuclei characteristic of apoptotic
cells could be found sporadically in wild-type tissues as well,
Journal of Cell Science
Apc6/Cdc16RNAi, UAS-Apc7RNAi or UAS-Apc8/Cdc23RNAi
constructs were collected and their lethal phase and mitotic
phenotypes were determined.
Similarly to single RNAi lines, the embryonic and early
larval developments were not affected in any of the double
RNAi combinations. However, as Table 1 illustrates, the late
larval lethality and the number of larvae with melanotic
tumours were greatly increased in the Apc6/Cdc16RNAiApc8/Cdc23RNAi (27%) and Apc7RNAi-Apc8/Cdc23RNAi (20%)
combinations compared to Apc6/Cdc16RNAi (6%), Apc7RNAi
(viable) or Apc8/Cdc23RNAi (13%) alone. The Apc6/Cdc16RNAiApc8/Cdc23RNAi and the Apc7RNAi-Apc8/Cdc23RNAi double
mutants also showed a definite shift in pupal lethality toward
earlier stages compared to the single RNAi lines (Table 1).
The range of mitotic phenotypes found in the
Apc6/Cdc16RNAi-Apc8/Cdc23RNAi
and
the
Apc7RNAiRNAi
Apc8/Cdc23
double RNAi lines was essentially the same
as that found in the Apc6/Cdc16RNAi and Apc8/Cdc23RNAi lines
alone; however, the MI of these double RNAi combinations
were significantly higher (4.4 and 4.2, respectively; P<0.05)
than that of Apc6/Cdc16RNAi (3.2), Apc7RNAi (2.1) or
Apc8/Cdc23RNAi (3.5) alone (Table 2). Taken together, the
increased L3 lethality, the shift of lethal phase and the more
Fig. 5. Cyclin B is not degraded
in RNAi silenced Apc6/Cdc16
and Apc8/Cdc23 cells. Colours
are the same as on Fig. 4. In wildtype cells (n=10) the cyclin B
level is high in prophase and
prometaphase (A-D) and it starts
to diminish at or after the onset of
metaphase (E-H). Cyclin B
staining is quite pronounced in
more than 80% of RNAi silenced
Apc6/Cdc16 (I-L) and
Apc8/Cdc23 (M-P) cells (n=20
and 17, respectively) arrested in
metaphase indicating that cyclin
B is not degraded.
TPR subunits of the APC/C have different functions
3245
Journal of Cell Science
accumulation was much more intensive in the Apc8/Cdc23
RNAi induced brains than in the control. The Apc6/Cdc16
preparations yielded similar results to Apc8/Cdc23.
The pycnotic nuclei appeared as doublets in neighbouring
cells (though their partition into two cells was not always
obvious in our preparations; Fig. 7A,B). This implied that the
induction of apoptosis occurred immediately after completing
mitosis in those cells that managed to escape the mitotic arrest.
Fig. 6. FLAG affinity chromatography of FLAG-tagged Apc7 and
Apc8 complexes. (A) Western blots of total protein extracts from S2
cells transfected with HA-tagged Apc8 (H8, lane 1) or FLAG-tagged
Apc7 (F7, lane 2) plasmids. Strip 1 was reacted with anti-HA (␣H,
lane 1), and strip 2 with anti-FLAG (␣F, lane 2) monoclonal
antibodies. (B) Western blots of proteins affinity-purified on antiFLAG-M2 antibody beads from S2 cells co-transfected with FLAGApc7 and HA-Apc8 (F7-H8, lanes 3 and 4), FLAG-Apc8 and HAApc7 (F8-H7, lanes 5 and 6) or FLAG-Apc7 and HA-Apc3 (F7-H3,
lanes 7 and 8) plasmids. Blots were reacted with anti-FLAG (lanes 3,
5 and 7) or anti-HA (lanes 4, 6 and 8) monoclonal antibodies. The
immunoreactive bands labelled with an asterisk are proteolytic
degradation products of Apc8. (C) Blots of anti-FLAG-M2 columnbound (lane 10) and unbound (flow-through, lane 9) proteins from
S2 cells transfected with HA-Apc8 (H8) plasmid and incubated with
anti-HA monoclonal antibody. Even after heavily overloading the
anti-FLAG column, no nonspecific binding of HA-Apc8 could be
detected. Similarly, HA-Apc3 did not bind to the anti-FLAG-M2
column (data not shown) E, eluate; FT, flow-through.
but their frequency increased by three- to fourfold in
Apc6/Cdc16 (P<0.005) and five to sixfold in Apc8/Cdc23
(P<0.005) knocked down preparations (Fig. 7E), suggesting
strong induction of apoptosis. To confirm that cell death was
induced in these RNAi lines, we stained larval brains with
Acridine Orange. As Fig. 7C,D illustrates, the Acridine Orange
Discussion
The APC/C is essential for the ubiquitin-dependent
degradation of cell cycle regulatory proteins. The complex
multisubunit structure of APC/C facilitates its intimate
involvement in the formation of substrate-ubiquitin conjugates,
and thus determines substrate specificity of the whole process.
More than half of the total mass of the APC/C consists of
tandem arrays of TPR motifs present in several evolutionarily
conserved subunits. Little is known about the specific functions
of the TPR subunits, although a structural role to form a
scaffold-like core of the APC/C has been suggested (Passmore
et al., 2005). The TPR subunits in budding yeast are coded by
the Cdc27, Cdc16 and Cdc23 genes that are all essential with
uniform loss-of-function mitotic arrest phenotypes. We show
here that in addition to the orthologues of these subunits, a gene
is present in the Drosophila genome that codes for a protein
closely related to the Apc7 subunit of the vertebrate APC/C.
Our data support the nomination of this protein as the
Drosophila Apc7 homologue. Firstly, its primary sequence
appears to be conserved, including the number, location and
distribution of its TPR motifs. The TPR motifs in the
Drosophila protein are more related to the TPR motifs of the
human Apc7 subunit than to the motifs of other Drosophila
TPR proteins, suggesting functional correspondence.
Secondly, the putative Apc7 protein is associated with
universal subunits of the Drosophila APC/C. Our affinity
purification data suggest that this association is either weak, or
occurs only in certain forms of the APC/C complex. The
existence of multiple forms of the APC/C has previously been
suggested (Huang and Raff, 2002), and APC/C dimers were
Fig. 7. Loss of both Apc6/Cdc16 and
Apc8/Cdc23 functions induces
apoptosis. Orcein stained preparations
from brains of Apc6/Cdc16 (A) and
Apc8/Cdc23 (B) RNAi L3 larvae show
small, rounded cells usually in pairs
with intense but uneven nucleolar
staining resembling apoptotic cells.
Acridine Orange staining further
indicates the high incidence of dying
cells in brains of RNAi larvae (D, only
an Apc8/Cdc23 RNAi sample is shown)
relative to wild-type larvae (C). (E) Bar
graph showing the apoptotic index in
neuroblast preparations of the
respective genotypes. The apoptotic
index is given as the mean number of
apoptotic cells in a microscope field.
Journal of Cell Science
3246
Journal of Cell Science 120 (18)
reported in budding yeast (Passmore et al., 2005).
Alternatively, it is also possible that Apc7 is not a core subunit
but a transiently interacting nonessential partner of the
Drosophila APC/C.
Thirdly, the putative Drosophila Apc7 gene shows
synergistic genetic interaction with the Apc8/Cdc23 gene that
also implicates a functional relationship. Although the idea was
that Apc7 is only a vertebrate-specific subunit of the APC/C
(Yu et al., 1998), it is now shown to be also present in species
from plants and insects, suggesting that this protein became
associated with the APC/C in most multicellular organisms.
We have previously shown that one of the TPR genes in
Drosophila, Apc3/Cdc27/mks, is essential and involved in both
cyclin A and cyclin B degradation (Deak et al., 2003). To
address the function of the other TPR proteins, we established
transgenic RNAi lines to reduce Apc6/Cdc16-, Apc7- and
Apc8/Cdc23-specific gene expression in intact animals.
Knocking down the expression of Apc6/Cdc16 or Apc8/Cdc23
genes caused lethality, indicating that as in yeast, these
genes are essential in Drosophila as well. Furthermore,
Apc6/Cdc16RNAi and Apc8/Cdc23RNAi cells show mitotic
phenotypes with high mitotic index, overcondensed
chromosomes and metaphase-like arrest that is very similar to
the mr, mks1 and ida mutants that code for the Apc2,
Apc3/Cdc27/Mks and Apc5 subunits of the Drosophila
APC/C, respectively (Reed and Orr-Weaver, 1997; Deak et al.,
2003; Bentley et al., 2002). This hallmark phenotype supports
the assumption that the Apc6/Cdc16 and Apc8/Cdc23 genes
code for genuine, functionally conserved APC/C subunits in
Drosophila.
Unlike in yeast, however, the phenotypes of the TPR mutants
are not uniform, since beyond common characteristics, they
show significant differences. Whereas the Apc7RNAi and Apc71
individuals are viable, mks1 mutants die as pharate adults,
Apc6/Cdc16RNAi animals die as P5(i) early pupae, and
Apc8/Cdc23RNAi animals die as P4(ii) prepupae. Differences
can also be seen in the ability to degrade APC/C substrates,
such as the mitotic cyclins. In this regard, removal of Apc7 has
no effect, depletion of Apc6/Cdc16 and Apc8/Cdc23 appear to
affect mainly cyclin B, whereas loss of Apc3/Cdc27/Mks
affects both cyclin A and B degradation. These observations
suggest different requirements of the TPR subunits in mitotic
cyclin degradation. However, it should be noted that these
differences could also be due to different inactivation
efficiencies, despite the fact that the residual expression levels
of the genes appear to be very similar in all TPR mutants as
judged by RT-PCR (Fig. 2B) (Deak et al., 2003). In this case
depletion of individual TPR subunits may affect the affinity of
substrate binding, or the efficiency of the ubiquitylation
reaction leading to an APC/C activity level at which cyclin A
degradation is still sufficient, whereas cyclin B degradation is
not.
The metaphase-like arrest of Apc6/Cdc16RNAi and
Apc8/Cdc23RNAi cells is unique in that they frequently show de
facto metaphase arrest with overcondensed chromosomes
aligned precisely at the metaphase plate. This correlates with
the ability of these cells to degrade cyclin A. This is in stark
contrast with mks1 cells that accumulate cyclin A and do not,
or seldom, form literal metaphase figures. These observations
suggest that cyclin A degradation is required in Drosophila for
proper chromosome alignment at metaphase. This is consistent
with the delay in chromosome alignment reported for the
overexpression of cyclin A in HeLa and PtK1 cells (den Elzen
and Pines, 2001) and overexpression of stable cyclin AS in
Drosophila embryos (Parry et al., 2003). In Apc6/Cdc16RNAi
and Apc8/Cdc23RNAi cells, degradation of cyclin A facilitates
chromosome alignment, whereas the imposed mitotic arrest
prolongs metaphase and hence the higher number of cells with
overcondensed but precisely aligned chromosomes. The
chromosomes of cells escaping from or passing this phase
become scattered similarly to mks1 arrested cells. This
appearance is the result of uncoordinated, oscillating poleward
movements of chromosomes caused by the accumulation of
cyclin B (Parry and O’Farrell, 2001). Since a high level of
cyclin A has a similar effect on chromosome movements in the
arrested cells as cyclin B (den Elzen and Pines, 2001), the
accumulation of both cyclin A and B prevents chromosome
alignment in mks1 cells.
The diverse phenotypic features of the TPR mutants are
somewhat surprising and appear to be inconsistent with the
predicted role of TPR subunits to form a central scaffold-like
structure to mediate APC/C assembly (Vodermaier et al., 2003;
Passmore et al., 2005). One would expect such a scaffold to
become unstable or collapse when its components are removed
one by one or in double mutant combinations, leading to
inactive APC/C, but we could not detect that in our RNAi lines.
Instead, all single and double TPRRNAi lines had slightly
smaller than normal larval brains and imaginal discs that
indicate reasonable mitotic activity and the existence of an
APC/C with at least residual functions. Since the TPR subunits
show extensive structural similarities, it is conceivable that a
missing subunit can be substituted by another one. This could
theoretically explain the relative stability of the scaffold
structure but it would not account for the different phenotypes
of the TPR subunits. Therefore, the data presented here better
fit an alternative model of APC/C that is centred on the largest
subunit, the Apc1/Shtd (Shattered in Drosophila), as a scaffold
(Thornton et al., 2006). In this model, the Apc1/Shtd provides
a platform to which all the other subunits bind by forming
functional subcomplexes. Consistently with this model, the
loss-of-function Apc1/shtd Drosophila mutants express an
extremely strong mitotic phenotype including larval lethality,
lack of imaginal discs, strong metaphase arrest with no or rare
anaphases, and very high frequency of polyploidy (M.P. and
P.D., unpublished). The mitotic phenotype of Apc1/shtd
mutants appears stronger than all the other known APC/C
subunit mutants, and RNAi lines presented here, suggesting
complete loss of APC/C structure and function. Taken all data
together, our view is that the TPR subunits may form functional
subcomplexes of the APC/C that could bind to the Apc1/Shtd
scaffold and most probably be involved in activator and
substrate binding.
Removal of most of the TPR subunits seems to have a direct
effect on cell viability as well. This is apparent from the
significant increase in the number of apoptotic cells in the
mitotically active larval brains of Apc6/Cdc16RNAi and
Apc8/Cdc23RNAi (Fig. 7), or mks1 animals (Deak et al., 2003).
These perturbations of APC/C function cause stabilization of
cyclin B, thus leading to prolonged activation of the mitotic
kinase, Cdc2, at the metaphase to anaphase progression and
beyond, when its activity must normally plummet to allow
mitosis to proceed. A plausible explanation for the high level
TPR subunits of the APC/C have different functions
of apoptosis is that the extended active state of Cdc2 during
late mitosis and mitotic exit could induce cell death. High Cdc2
activity has been found in apoptotic conditions and shown to
be required for the induction of this process (Fotedar et al.,
1995; Konishi et al., 2002). Since the molecular details of Cdc2
action are not fully known, further investigation is required to
link cell cycle regulation to the cell death machinery.
Materials and Methods
Fly stocks
Fly stocks were cultured at 25°C on standard Drosophila food. A w1118 isogenic
stock was used as wild type (Ryder et al., 2004). All genetic markers used are
described by Lindsley and Zimm (Lindsley and Zimm, 1992). Stocks were obtained
from the Bloomington Stock Center unless stated otherwise. For phenotypic
analysis, the chromosomes carrying a transgenic construct were balanced over CyO,
ActGFP or T(2;3)TSTL, CyO: TM6B, Tb.
The Apc71 mutant was established after imprecise excision of the
P{EPgy2}EY11333 element localized 63 bp downstream of the predicted stop
codon of CG14444 (Bellen et al., 2004). Five lines were recovered carrying internal
deletions of this gene. The largest deletion removed 1576 bp, about two-thirds of
the gene, starting at the insertion site and extending toward the 5⬘ end of the Apc7
gene. The insertion site is marked by a 24 bp long remnant of the original
P{EPgy2}EY11333 element. The resulting truncated Apc7 protein lacks two-thirds
of its mass including the eight tandem TPR repeats. This mutation was designated
as Apc71 and used in this work as a knocked out allele of the Apc7 gene.
Journal of Cell Science
Transgenic RNA-interference lines
To make transgenic double-stranded RNA-interference constructs, PCR primer pairs
of 5⬘-ACTCTAGATTGCCTGTCTGGTGGAAAAC-3⬘ and 5⬘-ATTCTAGACTTGCGCAGCGAATGACC-3⬘; 5⬘-ACTCTAGAGCTCTCGCCCAAATGTTCA-3⬘
and 5⬘-ATTCTAGAGAGGCGTATCGTCCAGTTGC-3⬘; and 5⬘-ACTCTAGAACGCGCCCTGAAACTGAAT-3⬘ and 5⬘-ATTCTAGATCGACGCATGCCCTGAAT3⬘ were used to amplify 656 bp, 917 bp and 910 bp sequences corresponding to
exon 5, exon 3 and exon 2 and 3 of the Drosophila Cdc16, Apc7 and Cdc23
transcripts, respectively (XbaI sites are underlined). The PCR products were cloned
into the pWIZ vector (a gift from Richard Carthew, Northwestern University, USA),
in opposite orientation on both sides of the white intron present in the vector (Lee
and Carthew, 2003). The obtained gene-specific RNAi constructs were designated
as P{UST-Cdc16RNAi}, P{UST-Apc7RNAi} and P{UST-Cdc23RNAi}, and all
constructs were verified before being processed for transformation. Following Pelement-mediated germline transformation (Spradling and Rubin, 1982) of w1118
isogenic flies, at least five independent transgenic lines were obtained for each gene.
To test RNAi effects, homozygous transgenic flies carrying subunit-specific RNAi
constructs (such as P{UST-Cdc16RNAi}) were crossed to Act5C-GAL4 females
which expressed the Gal4 transcription factor under the control of the actin5C
promoter. The progeny of these crosses were examined for subunit-specific gene
expression as well as lethal and mitotic phenotypes.
RT-PCR
Total RNA was isolated from young pupae using the Tri Reagent extraction kit
(Sigma). The RNA samples were DNase treated with RQ1 RNase-Free DNase
(Promega). cDNA was synthesized from 5 ␮g total RNA as template with M-MuLV
reverse transcriptase (Fermentas, Vilnius, Lithuania) and random hexanucleotide
primers (Fermentas, Vilnius, Lithuania). Two primers complementary to the 4. and
5. exons of Apc6, to the 2. and 3. exons of Apc7 and to the 1. and 3. exons of Apc8
were used in 20 cycle PCR amplifications. 20 cycle PCRs were performed using
rpL17A primers (rpL17A upper, 5⬘-GTGATGAACTGTGCCGACAA-3⬘; rpL17A
lower, 5⬘-CCTTCATTTCGCCCTTGTTG-3⬘) on the same cDNA templates to serve
as calibration controls. RT-PCR products were separated by agarose gel
electrophoresis.
Cytology
Brains from third instar larvae were dissected in a large drop of 0.7% NaCl solution
then transferred to a drop of 45% acetic acid on a microscope slide. After 10-15
seconds, the liquid was absorbed with a piece of filter paper and replaced with a
drop of 3% orcein (Gurr’s 23282) in 45% acetic acid. After 3-5 minutes staining,
the orcein was removed and the brains were destained in a drop of 65% acetic acid
for 5-10 seconds. Following that, the destaining solution was replaced with a drop
of 3% orcein in 65% acetic acid. The drop was immediately covered with a
coverslip, wrapped up in filter paper and squashed firmly for 10-15 seconds. The
preparations were examined and photographed under phase-contrast optics using an
Olympus BX51 upright microscope with a UPLFLN 100XO2PH plan-apochromat
objective. A DP 70 digital colour camera was used to take images. Mitotic index
(MI) was defined as the number of mitotic cells on an optical field. On average, 1020 optical fields per preparation were scored. The mean number of cells at different
3247
stages of mitosis was determined for each single or double mutant line and
compared using Student’s t-test.
Quantification of cell death was performed by observing the morphology of
orcein-stained cells, and scoring for apoptotic cells with pycnotic nuclei. The mean
number of apoptotic cells was determined and used for further statistical analysis.
Acridine Orange staining was performed by dissecting third instar larval brains in
a drop of PBS. The brains were transferred into a drop of 1.6 ␮g/ml Acridine Orange
(C.I. 46005, Molar Chemicals) solution and incubated for 5 minutes in the dark,
then rinsed in PBS. Following that, the brains were transferred to a drop of PBS on
a microscope slide, covered with a coverslip and sealed using nail polish. Before
transferring the brains, two Sellotape cushion were made on the slides. The
preparations were examined using an Olympus FV1000 confocal microscope within
15-20 minutes from dissection.
Epitope constructs and affinity chromatography
Plasmid constructs to express N-terminal FLAG and haemagglutinin (HA)
epitope-tagged proteins in S2 cells under the control of the Actin-5C promoter
were constructed using the Drosophila Gateway Vector System
(http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html). First, full-length
cDNAs of Apc3/Cdc27/mks, Apc7 and Apc8/Cdc23 were cloned into the appropriate
entry (pENTR) vectors in frame. Following that, the ORFs of these genes were
recombined either into the pAFW or pAHW destination vectors using Gateway LR
clonase II Enzyme Mix (Invitrogen). The pAFW vector contains three FLAG
whereas the pAHW vector contains three HA epitope tags 5⬘ of the Gateway
cassette. Successful recombinant clones were selected based on their resistance to
ampicillin and lack of ccdB toxicity. All clones were verified by sequencing before
transfection. The N-terminal fusion constructs were designated in this paper as
follows: pAFW-Apc7 as FLAG-Apc7 or F7; pAFW-Apc8 as FLAG-Apc8 or F8;
pAHW-Apc3 as HA-Apc3 or H3; pAHW-Apc7 as HA-Apc7 or H7 and pAHWApc8 as HA-Apc8 or H8.
Schneider 2 (S2) tissue culture cells were either transfected with 2.5 ␮g plasmid
DNA of FLAG-Apc7, HA-Apc7, HA-Apc3, FLAG-Apc8 or HA-Apc8 alone or cotransfected with 2.5+2.5 ␮g FLAG-Apc7+HA-Apc3, FLAG-Apc7+HA-Apc8 or
FLAG-Apc8+HA-Apc7 double combinations using 15 ␮l Cellfectin (Invitrogen) in
serum-free S2 cell medium (Sigma). For preparation of total protein extracts,
transfected cells were dissolved in SDS sample buffer 48 hours after transfection.
For affinity chromatography, cells were washed in PBS 48 hours after transfection
and cell extracts were prepared by incubating the cells in 25 mM Hepes pH 7.6,
10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and 0.2%
NP40. After 10 minutes incubation on ice, an equal volume of cytoplasmic dilution
buffer (25 mM Hepes pH 7.6, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM
EGTA, 1 mM PMSF and 450 mM NaCl) was added, the nuclei were removed by
centrifugation (3 minutes at 10,000 g) and FLAG-tagged proteins were affinitypurified on anti-FLAG-M2-agarose beads (Sigma-Aldrich). After 2 hours of
incubation at 4°C, anti-FLAG-M2-agarose beads were washed four times with five
volumes of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and bound proteins were eluted
with an excess of FLAG peptides. Total protein extracts or affinity-purified samples
were fractionated on 8% SDS-PAGE and blotted to PVDF membrane strips. Western
blots were reacted with anti-FLAG or anti-HA monoclonal antibodies.
Immunostaining
Third instar larval brains (n=3-6) were dissected in PBS, fixed in ice-cold methanol
for 20 minutes, washed three times for 5 minutes in PBS and than permeabilised in
PBS containing 0.3% Triton X-100 for 10 minutes at room temperature and washed
again in PBS containing 1% BSA for 40 minutes at room temperature. The brains
were incubated with primary antibodies diluted to an appropriate concentration in
PBS containing 1% BSA overnight in a humid chamber. They were than washed
four times for 15 minutes each with PBST (PBS supplemented with 0.1% Triton
X-100) containing 1% BSA at room temperature before being incubated with
appropriate secondary antibodies (Jackson) diluted in PBST containing 1% BSA
for 2 hours at room temperature in the dark. The preparations were finally washed
twice for 15 minutes in PBST and two times 15 minutes in PBS. DNA was stained
with TOTO-3 (Molecular Probes) before the specimens were mounted in
Vectashield.
The primary antibodies were Rb270 rabbit anti-cyclin A antibody (Whitfield et
al., 1990), Rb271 rabbit anti-cyclin B antibody (Whitfield et al., 1990) and rat
monoclonal anti-␣-tubulin antibody, YL1/2 (Kilmartin at al., 1982). The
preparations were examined using an Olympus FV1000 confocal microscope. For
comparing cyclin levels, images of metaphase cells were acquired using identical
settings and cyclin immunostaining was determined by visual inspection of the
images.
We wish to thank Katalin Udvardy for her contribution in carrying
out the S2 cell transfection experiments. We are grateful to Mary Lilly
and Edward Wojcik for discussions and comments on the manuscript.
This work was supported by a Hungarian Scientific Research Fund
(OTKA) grant awarded to P.D. (T037322).
3248
Journal of Cell Science 120 (18)
Journal of Cell Science
References
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and
Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res. 25, 3389-3402.
Bainbridge, S. P. and Bownes, M. (1981). Staging the metamorphosis of Drosophila
melanogaster. J. Embryol. Exp. Morphol. 66, 57-80.
Bellen, H. J., Levis, R. W., Liao, G., He, Y., Carlson, J. W., Tsang, G., Evans-Holm,
M., Hiesinger, P. R., Schulze, K. L., Rubin, G. M. et al. (2004). The BDGP gene
disruption project: single transposon insertions associated with 40% of Drosophila
genes. Genetics 167, 761-781.
Bentley, A. M., Williams, B. C., Goldberg, M. L. and Andres, A. J. (2002). Phenotypic
characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle
progression. J. Cell Sci. 115, 949-961.
Blatch, G. L. and Lässle, M. (1999). The tetratricopeptide repeat: a structural motif
mediating protein-protein interactions. BioEssays 21, 932-939.
Carroll, C. W. and Morgan, D. O. (2002). The Doc1 subunit is a processivity factor for
the anaphase-promoting complex. Nat. Cell Biol. 4, 880-887.
Das, A. K., Cohen, P. W. and Barford, D. (1998). The structure of the tetratricopeptide
repeats of protein phosphatase 5: implications for TPR-mediated protein-protein
interactions. EMBO J. 17, 1192-1199.
Deak, P., Donaldson, M. and Glover, D. M. (2003). Mutations in makos, a Drosophila
gene encoding the Cdc27 subunit of the anaphase promoting complex, enhance
centrosomal defects in polo and are suppressed by mutations in twins/aar, which
encodes a regulatory subunit of PP2A. J. Cell Sci. 116, 4147-4158.
den Elzen, N. and Pines, J. (2001). Cyclin A is destroyed in prometaphase and can delay
chromosome alignment and anaphase. J. Cell Biol. 153, 121-136.
Fotedar, R., Flatt, J., Gupta, S., Margolis, R. L., Fitzgerald, P., Messier, H. and
Fotedar, A. (1995). Activation-induced T-cell death is cell cycle dependent and
regulated by cyclin B. Mol. Cell. Biol. 15, 932-942.
Gatti, M. and Baker, B. S. (1989). Genes controlling essential cell-cycle functions in
Drosophila melanogaster. Genes Dev. 3, 438-453.
Gmachl, M., Gieffers, C., Podtelejnikov, A. V., Mann, M. and Peters, J. M. (2000).
The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to
ubiquitinate substrates of the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA
97, 8973-8978.
Harper, J. W., Burton, J. L. and Solomon, M. J. (2002). The anaphase-promoting
complex: it’s not just for mitosis any more. Genes Dev. 16, 2179-2206.
Huang, J. Y. and Raff, J. W. (2002). The dynamic localisation of the Drosophila APC/C:
evidence for the existence of multiple complexes that perform distinct functions and
are differentially localised. J. Cell Sci. 115, 2847-2856.
Kilmartin, J. V., Wright, B. and Milstein, C. (1982). Rat monoclonal antitubulin
antibodies derived by using a new nonsecreting rat cell line. J. Cell Biol. 93, 576582.
Konishi, Y., Lehtinen, M., Donovan, N. and Bonni, A. (2002). Cdc2 phosphorylation
of BAD links the cell cycle to the cell death machinery. Mol. Cell 9, 1005-1016.
Lamb, J. R., Michaud, W. A., Sikorski, R. S. and Hieter, P. A. (1994). Cdc16p, Cdc23p
and Cdc27p form a complex essential for mitosis. EMBO J. 13, 4321-4328.
Lamb, J. R., Tugendreich, S. and Hieter, P. (1995). Tetratrico peptide repeat
interactions: to TPR or not to TPR? Trends Biochem. Sci. 20, 257-259.
Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophila melanogaster. San
Diego: Academic Press.
Lee, Y. S. and Carthew, R. W. (2003). Making a better RNAi vector for Drosophila: use
of intron spacers. Methods 30, 322-329.
Leverson, J. D., Joazeiro, C. A., Page, A. M., Huang, H., Hieter, P. and Hunter, T.
(2000). The APC11 RING-H2 finger mediates E2-dependent ubiquitination. Mol. Biol.
Cell 11, 2315-2325.
Ohta, T., Michel, J. J., Schottelius, A. J. and Xiong, Y. (1999). ROC1, a homolog of
APC11, represents a family of cullin partners with an associated ubiquitin ligase
activity. Mol. Cell 3, 535-541.
Parry, D. H. and O’Farrell, P. H. (2001). The schedule of destruction of three mitotic
cyclins can dictate the timing of events during exit from mitosis. Curr. Biol. 11, 671683.
Parry, D. H., Hickson, G. R. and O’Farrell, P. H. (2003). Cyclin B destruction triggers
changes in kinetochore behavior essential for successful anaphase. Curr. Biol. 13, 647653.
Passmore, L. A. (2004). The anaphase-promoting complex (APC): the sum of its parts?
Biochem. Soc. Trans. 32, 724-727.
Passmore, L. A., Booth, C. R., Venien-Bryan, C., Ludtke, S. J., Fioretto, C., Johnson,
L. N., Chiu, W. and Barford, D. (2005). Structural analysis of the anaphase-promoting
complex reveals multiple active sites and insights into polyubiquitylation. Mol. Cell
20, 855-866.
Reed, B. H. and Orr-Weaver, T. L. (1997). The Drosophila gene morula inhibits mitotic
functions in the endo cell cycle and the mitotic cell cycle. Development 124, 35433553.
Ryder, E., Blows, F., Ashburner, M., Bautista-Llacer, R., Coulson, D., Drummond,
J., Webster, J., Gubb, D., Gunton, N., Johnson, G. et al. (2004). The DrosDel
collection: a set of P-element insertions for generating custom chromosomal
aberrations in Drosophila melanogaster. Genetics 167, 797-813.
Spradling, A. C. and Rubin, G. M. (1982). Transposition of cloned P elements into
Drosophila germ line chromosomes. Science 218, 341-347.
Tang, Z., Li, B., Bharadwaj, R., Zhu, H., Ozkan, E., Hakala, K., Deisenhofer, J. and
Yu, H. (2001). APC2 Cullin protein and APC11 RING protein comprise the minimal
ubiquitin ligase module of the anaphase-promoting complex. Mol. Biol. Cell 12, 38393851.
The FlyBase Consortium (2003). The FlyBase database of the Drosophila genome
projects and community literature. Nucleic Acids Res. 31, 172-175.
Thornton, B. R., Ng, T. M., Matyskiela, M. E., Carroll, C. W., Morgan, D. O. and
Toczyski, D. P. (2006). An architectural map of the anaphase-promoting complex.
Genes Dev. 20, 449-460.
Vodermaier, H. C., Gieffers, C., Maurer-Stroh, S., Eisenhaber, F. and Peters, J. M.
(2003). TPR subunits of the anaphase-promoting complex mediate binding to the
activator protein CDH1. Curr. Biol. 13, 1459-1468.
Whitfield, W. G., Gonzalez, C., Maldonado-Codina, G. and Glover, D. M. (1990). The
A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct
events that define separable phases of the G2-M transition. EMBO J. 9, 2563-2572.
Yu, H., Peters, J. M., King, R. W., Page, A. M., Hieter, P. and Kirschner, M. W. (1998).
Identification of a cullin homology region in a subunit of the anaphase-promoting
complex. Science 279, 1219-1222.