We have cloned and characterized the ida gene that is required for proliferation of imaginal disc cells during Drosophila development. IDA is homologous to APC5, a subunit of the anaphase-promoting complex(APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of IDA is required for the production of eggs and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, as mitotic stages in which chromosomes are orientated at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex.

Introduction

In holometabolous insects such as Drosophila, larval tissues are set aside from adult or imaginal tissues during early embryogenesis(Bate and Arias, 1991). During the instars, larval cells become polytenized and grow in size without cell division, and most will undergo a steroid-hormone-regulated programmed cell death at metamorphosis (Jiang et al.,1997). By contrast, cells in the imaginal tissues proliferate and are maintained in a strictly diploid undifferentiated state throughout larval development. At metamorphosis, it is the imaginal cells that will differentiate to form the adult body plan. Because of these fundamental differences in cell fate, and because a maternal contribution is often sufficient to sustain all embryonic cell divisions, mutations in many genes regulating the diploid cell cycle do not cause lethality until metamorphosis. However, homozygous mutants eventually die because imaginal disc structures are not present to replace the histolyzing larval tissues. By examining mutants that display late-larval or prepupal lethal periods, many genes have been identified that play a role in regulating the diploid cell cycle(Sunkel and Glover, 1988;Gatti and Baker, 1989;Treisman et al., 1995;Basu et al., 1999). Thus, it is logical to predict that mutations in subunits of the anaphase-promoting complex would also display such phenotypes.

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.

Materials and Methods

Drosophila stocks and mutant analysis

All flies were reared on standard cornmeal-molasses medium supplemented with baker's yeast. Stocks were obtained from the Bloomington Stock Center(Bloomington, IN) unless otherwise noted. Wild-type samples are from Canton S or Oregon R strains. The idab4 [l(3)63FbB4] and idad14 [l(3)63FbD14] alleles were kindly provided by Stephen Harrison (Harrison et al., 1995). The esg-lacZ insertion stock[P[PZ]esg05729 cn/CyO](Hayashi et al., 1993) was provided by the Berkeley Drosophila Genome Project (Berkeley, CA)through Todd Laverty. The deficiency stocks Df(3L)449 and Df(3L)107 (idaΔ107), and the P[w+, 63F/k1] transgenic line were generated in a previous study (Vaskova et al., 2000). The idak1-449 synthetic deficiency{P[w+63/k1]; Df(3L)449/TM6B}was generated by placing mge genomic DNA{P[w+, 63F/k1]} into the Df(3L)449 stock containing a deletion of mge and ida loci. For phenotypic analysis, deficiencies and idamutants were balanced over TM6B, Tb e Hu, and Tb+ progeny from stocks and/or crosses were selected for analysis.

Cloning ida and characterizing mutant alleles

Total RNA (20 μg) from wild-type and idaΔ107third-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 idaΔ107 animals was prepared by random labeling(Prime It II Kit, Stratagene) and used to screen a 0-24 hour λZAPII 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 idamutant 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 Δ107, 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, Sbmales. 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

β-galactosidase activity from the esg-lacZ line was detected in dissected larval tissues using X-gal (5-bromo-4-chloro-3-indolylβ-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-α-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 1×106 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.

Results

ida mutants die during the prepupal period and lack mature imaginal disc structures

In a previous genetic analysis, mutations defining the ida gene were mapped to the 63F3-7 region of chromosome 3(Vaskova et al., 2000). These non-complementing mutations include a ∼2.7-kb deficiency generated by P-element transposition and excision (idaΔ107), and two EMS-derived mutant alleles (idab4 and idad14) from a previously identified but uncharacterized genetic locus (Harrison et al.,1995). Animals homozygous for idad14,idad14, or idaΔ107, and hemizygous with any of these alleles over Df(3L)GN19 (a large deletion of the 63F region), display identical prepupallethal phenotypes, suggesting that the idaΔ107 deficiency and the two EMS-induced mutations behave genetically as null alleles. In these assays, the synthetic deficiency stock, idak1-449, also behaves as a genetic null even though it may produce some functional protein (see below).

Though embryonic and larval development is normal(Vaskova et al., 2000), all ida mutants reported here (idaΔ107,idab4, idad14 and idak1-449)stall in metamorphic development between 3 and 6 hours after puparium formation (APF). Fig. 1A,Bcompares 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.

Fig. 1.

ida mutants die during metamorphosis and are `imaginal discs arrested'. Wild-type (A) and idaΔ107 (B) animals are pictured at 48 hours after puparium formation. The arrow marks the position of a stalled gas bubble (B). β-gal activity is detected in dissected tissues from esg-lacZ; idaΔ107/+ (C) and esg-lacZ;idaΔ107/idaΔ107 (D) wandering third-instar larvae. Arrows point to proliferation zones in the optic lobes that are reduced in size in ida mutants (D). Arrowheads mark the position of excorporate imaginal discs in (C), and rudimentary imaginal discs in (D). Hoechst staining of the anterior gut in wild-type (E) and idaΔ107 (F) larvae is shown. An arrow marks the presence (E) and absence (F) of the imaginal ring. β-gal staining in the anterior gut of an esg-lacZ; idaΔ107/+ (G) and an esg-lacZ; idaΔ107/idaΔ107 (H)animal is presented. Note the severe reduction of β-gal positive cells(arrows, H) in the imaginal ring in idaΔ107 animals.β-gal staining in the anterior dorsal (ad) and posterior dorsal (pd)histoblast nests from esg-lacZ; idaΔ107/+ (I) and esg-lacZ; idaΔ107/idaΔ107 (J)animals are indicated. Adjacent segments are presented for each genotype. No differences are detected in primordial cell number.

Fig. 1.

ida mutants die during metamorphosis and are `imaginal discs arrested'. Wild-type (A) and idaΔ107 (B) animals are pictured at 48 hours after puparium formation. The arrow marks the position of a stalled gas bubble (B). β-gal activity is detected in dissected tissues from esg-lacZ; idaΔ107/+ (C) and esg-lacZ;idaΔ107/idaΔ107 (D) wandering third-instar larvae. Arrows point to proliferation zones in the optic lobes that are reduced in size in ida mutants (D). Arrowheads mark the position of excorporate imaginal discs in (C), and rudimentary imaginal discs in (D). Hoechst staining of the anterior gut in wild-type (E) and idaΔ107 (F) larvae is shown. An arrow marks the presence (E) and absence (F) of the imaginal ring. β-gal staining in the anterior gut of an esg-lacZ; idaΔ107/+ (G) and an esg-lacZ; idaΔ107/idaΔ107 (H)animal is presented. Note the severe reduction of β-gal positive cells(arrows, H) in the imaginal ring in idaΔ107 animals.β-gal staining in the anterior dorsal (ad) and posterior dorsal (pd)histoblast nests from esg-lacZ; idaΔ107/+ (I) and esg-lacZ; idaΔ107/idaΔ107 (J)animals are indicated. Adjacent segments are presented for each genotype. No differences are detected in primordial cell number.

To determine the cause of lethality, we dissected idaΔ107, idab4, idad14 and idak1-449 homozygous wandering third-instar larvae. Data for the idaΔ107 deficiency is presented here,although all homozygous and hemizygous null mutants display identical phenotypes. Brightfield images of dissected tissue show that idamutants 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 forβ-galactosidase (β-gal) activity in wandering third-instar larval stage. In control larvae heterozygous for ida, β-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 β-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 inFig. 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 β-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 idaphenotype, 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 idaΔ107 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-449animals (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.

Fig. 2.

A single 2.3 kb mRNA is disrupted in ida mutants. (A) A schematic of the 63F genetic locus is presented. The position of three subclones from the 63F walk and dSc2, ida, and mge gene structures are shown. Exons are demarcated by open rectangles, and small arrows mark the direction of transcription. Genomic DNA missing in idaΔ107 and Df(3L)449 deficiency stocks is indicated by gaps marked with parenthesis. A solid line represents genomic DNA present in the P[w+63F/k1] transgene(see text for details). The proximal boundary of the P[w+63F/k1] transgene is off the scale of the figure. (B) Three identical northern blots with samples isolated from homozygous animals of the genotypes indicated were prepared. The probes used are designated above each blot. A strand-specific riboprobe generated against the full-length p63F.15 subclone using T3 polymerase hybridizes to ida and dSc2 transcripts. A riboprobe generated using T7 polymerase recognizes mge. Note that mge and dSc2transcript levels are unaffected in ida mutants. A blot hybridized for rp49 serves as a loading and blotting control.

Fig. 2.

A single 2.3 kb mRNA is disrupted in ida mutants. (A) A schematic of the 63F genetic locus is presented. The position of three subclones from the 63F walk and dSc2, ida, and mge gene structures are shown. Exons are demarcated by open rectangles, and small arrows mark the direction of transcription. Genomic DNA missing in idaΔ107 and Df(3L)449 deficiency stocks is indicated by gaps marked with parenthesis. A solid line represents genomic DNA present in the P[w+63F/k1] transgene(see text for details). The proximal boundary of the P[w+63F/k1] transgene is off the scale of the figure. (B) Three identical northern blots with samples isolated from homozygous animals of the genotypes indicated were prepared. The probes used are designated above each blot. A strand-specific riboprobe generated against the full-length p63F.15 subclone using T3 polymerase hybridizes to ida and dSc2 transcripts. A riboprobe generated using T7 polymerase recognizes mge. Note that mge and dSc2transcript levels are unaffected in ida mutants. A blot hybridized for rp49 serves as a loading and blotting control.

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 idaΔ107 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 idacDNA, 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.

Fig. 3.

ida encodes an APC5 homolog. (A) IDA coding information is in uppercase letters. 5′-untranslated, 3′-untranslated, and the DNA for the single intron are in lowercase letters. Amino acid residues are in three-letter code. Numbers to the right represent nucleotide position (upper)relative to the end of dSc2, and amino acid position (lower) relative to the putative IDA start. The presumed polyA signal is bold and underlined. A blue box outlines the putative TPR domain. A pink box demarcates putative phosphorylation sites that are lost in the nonsense mutation of the idab4 allele (asterisk, see text for details). The GenBank accession number for ida is AF312026. (B) An alignment of putative TPR domains in APC5 homologs from Drosophila, H. sapiens, S. cerevisiae and C. elegans is presented. Red residues are identical in all species, orange residues indicate that at least two species share identity, and yellow indicates similar residues.

Fig. 3.

ida encodes an APC5 homolog. (A) IDA coding information is in uppercase letters. 5′-untranslated, 3′-untranslated, and the DNA for the single intron are in lowercase letters. Amino acid residues are in three-letter code. Numbers to the right represent nucleotide position (upper)relative to the end of dSc2, and amino acid position (lower) relative to the putative IDA start. The presumed polyA signal is bold and underlined. A blue box outlines the putative TPR domain. A pink box demarcates putative phosphorylation sites that are lost in the nonsense mutation of the idab4 allele (asterisk, see text for details). The GenBank accession number for ida is AF312026. (B) An alignment of putative TPR domains in APC5 homologs from Drosophila, H. sapiens, S. cerevisiae and C. elegans is presented. Red residues are identical in all species, orange residues indicate that at least two species share identity, and yellow indicates similar residues.

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 (CAG→TAG),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 byFoe 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 ovoD1female-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 idaalleles 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).

Table 1.

idamaternal is required for the production of viable embryos

Genotype of female parentHeat shockEggs laidResults.
hsFlp; D FRT/ovoD1 FRT No ovoD1/ovo+ germlines produce no eggs 
hsFlp; D FRT/ovoD1 FRT Yes 4558 FLP induces recombination to generate ovo+/ovo+germlines that produce many eggs 
hsFlp; ida▵107 FRT/ovoD1 FRT Yes ida▵107/ida▵107 germlines produce very few eggs; all eggs fail to hatch 
hsFlp; idab4 FRT/ovoD1 FRT Yes idab4/idab4 germlines produce very few eggs;all eggs fail to hatch 
hsFlp; idad14 FRT/ovoD1 FRT Yes idad14/idad14 germlines produce very few eggs;all eggs fail to hatch 
Genotype of female parentHeat shockEggs laidResults.
hsFlp; D FRT/ovoD1 FRT No ovoD1/ovo+ germlines produce no eggs 
hsFlp; D FRT/ovoD1 FRT Yes 4558 FLP induces recombination to generate ovo+/ovo+germlines that produce many eggs 
hsFlp; ida▵107 FRT/ovoD1 FRT Yes ida▵107/ida▵107 germlines produce very few eggs; all eggs fail to hatch 
hsFlp; idab4 FRT/ovoD1 FRT Yes idab4/idab4 germlines produce very few eggs;all eggs fail to hatch 
hsFlp; idad14 FRT/ovoD1 FRT Yes idad14/idad14 germlines produce very few eggs;all eggs fail to hatch 

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.

Table 2.

The mitotic index is increased in ida mutant brains

GenotypeFields/brainCells in prometaphase*Cells in anaphaseMitotic index
Wildtype (Canton S) 83 36 14 0.6 
idaΔ107/idaΔ107 68 118 21 2.0 
idad14/idad14 73 116 30 2.0 
GenotypeFields/brainCells in prometaphase*Cells in anaphaseMitotic index
Wildtype (Canton S) 83 36 14 0.6 
idaΔ107/idaΔ107 68 118 21 2.0 
idad14/idad14 73 116 30 2.0 
*

Numbers include cells scored as both prometaphase and metaphase in wildtype squashes.

The index is calculated as the total number of cells with condensed chromosomes over the total number of fields examined.

Fig. 4.

ida mutants display abnormal chromosome cytology. Mitotic figures of wild-type prometaphase (A), wild-type anaphase (B), idaΔ107 prometaphase (C), and idaΔ107 anaphase (D) are presented. Prometaphase figures show that ida cells contain an increased number of chromatin structures (compare A with C). Aberrant anaphase figures with lagging chromosomes are observed in ida cells (arrow, D). Wild-type (E) and idaΔ107 (F) cells show normal staining patterns for tubulin (white) and centrosomin (green). Hoechst staining (blue) shows the position of chromosomes along the spindle.

Fig. 4.

ida mutants display abnormal chromosome cytology. Mitotic figures of wild-type prometaphase (A), wild-type anaphase (B), idaΔ107 prometaphase (C), and idaΔ107 anaphase (D) are presented. Prometaphase figures show that ida cells contain an increased number of chromatin structures (compare A with C). Aberrant anaphase figures with lagging chromosomes are observed in ida cells (arrow, D). Wild-type (E) and idaΔ107 (F) cells show normal staining patterns for tubulin (white) and centrosomin (green). Hoechst staining (blue) shows the position of chromosomes along the spindle.

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 idamutant 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 inFig. 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 idamutants.

Fig. 5.

ida cells are aneuploid. Mitotic figures from idak1-449 show examples of aneuploidy, with one cell containing 21 unfragmented chromosomes (A), and another containing 39 (B). Aneuploidy is also observed in null ida alleles. All individual Hoechst-labeled chromatin figures in an idaΔ107 cell(C) have a corresponding Bub1 antibody signal (D). Note that some of the Bub1 staining is not in the plane of focus.

Fig. 5.

ida cells are aneuploid. Mitotic figures from idak1-449 show examples of aneuploidy, with one cell containing 21 unfragmented chromosomes (A), and another containing 39 (B). Aneuploidy is also observed in null ida alleles. All individual Hoechst-labeled chromatin figures in an idaΔ107 cell(C) have a corresponding Bub1 antibody signal (D). Note that some of the Bub1 staining is not in the plane of focus.

Upstream spindle checkpoint events are active in idacells

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 idamutant 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).

Fig. 6.

Bub1 is detected at ida kinetochores throughout mitosis. Hoechst staining (A-C) shows chromosome morphology and Bub1 staining (D-F) marks kinetochore positions. In all panels, asterisks mark prometaphase, arrows mark metaphase, and brackets mark anaphase. Two fields of idaΔ107 cells are shown (B,C,E,F). Bub1 staining is present on prometaphase figures from both wild-type (D) and idaΔ107 cells (E,F). During anaphase, Bub1 staining decreases in wild-type (bracket, D). The staining intensity is the same at both stages in idaΔ107 cells (brackets, E,F). Note that some ida mutant cells are outside the plane of focus.

Fig. 6.

Bub1 is detected at ida kinetochores throughout mitosis. Hoechst staining (A-C) shows chromosome morphology and Bub1 staining (D-F) marks kinetochore positions. In all panels, asterisks mark prometaphase, arrows mark metaphase, and brackets mark anaphase. Two fields of idaΔ107 cells are shown (B,C,E,F). Bub1 staining is present on prometaphase figures from both wild-type (D) and idaΔ107 cells (E,F). During anaphase, Bub1 staining decreases in wild-type (bracket, D). The staining intensity is the same at both stages in idaΔ107 cells (brackets, E,F). Note that some ida mutant cells are outside the plane of focus.

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 idamutants 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 byCarthew, 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 idaresults 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.

Fig. 7.

Sister-chromatid separation occurs in cells depleted of IDA. Sister-chromatid separation is observed in brain squashes from an idak1-449 mutant in which chromosomes are not hypercondensed (arrows, A). Hoechst staining of untreated Kc cells (B) is compared with cells treated with dsRNA specific for ida (C). Control cells retain normal chromosome morphology (B), while RNAi treated cells contain chromosomes with hypercondensed morphology and separated sister chromatids (arrowheads, C). A rarely occurring four-lobed structure, or unseparated chromatid pair is designated by an arrow (C). RNAi phenocopies the chromosome hypercondensation observed in ida null mutants.

Fig. 7.

Sister-chromatid separation occurs in cells depleted of IDA. Sister-chromatid separation is observed in brain squashes from an idak1-449 mutant in which chromosomes are not hypercondensed (arrows, A). Hoechst staining of untreated Kc cells (B) is compared with cells treated with dsRNA specific for ida (C). Control cells retain normal chromosome morphology (B), while RNAi treated cells contain chromosomes with hypercondensed morphology and separated sister chromatids (arrowheads, C). A rarely occurring four-lobed structure, or unseparated chromatid pair is designated by an arrow (C). RNAi phenocopies the chromosome hypercondensation observed in ida null mutants.

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 CDC27APC/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.

Fig. 8.

Cyclin B levels remain high in ida cells with anaphase chromosome positioning. Hoechst staining (A-D) and cyclin B staining (E-H) in wild-type(A,E,C,G), and idaΔ107 (B,F,D,H) cells is presented. In wild-type, cyclin B levels are high at prometaphase (E), but are reduced at anaphase (G). idaΔ107 mutant cells contain high cyclin B levels during prometaphase (F) in a pattern similar to wild-type. High cyclin B levels are retained during anaphase in most idaΔ107 cells (H).

Fig. 8.

Cyclin B levels remain high in ida cells with anaphase chromosome positioning. Hoechst staining (A-D) and cyclin B staining (E-H) in wild-type(A,E,C,G), and idaΔ107 (B,F,D,H) cells is presented. In wild-type, cyclin B levels are high at prometaphase (E), but are reduced at anaphase (G). idaΔ107 mutant cells contain high cyclin B levels during prometaphase (F) in a pattern similar to wild-type. High cyclin B levels are retained during anaphase in most idaΔ107 cells (H).

Table 3.

Cyclin B levels remain high during anaphase in idamutants

GenotypeHigh cyclin B at prometaphaseHigh cyclin B at metaphaseHigh cyclin B at anaphaseTotal mitotic cells scored
Wildtype (Canton S) 89% (59) 82% (23) 5% (18) 100 
idaΔ107/idaΔ107 96% (95) NA 84% (50) 145 
Parenthetical values are the number of cells scored for each stage. NA, ida cells do not contain metaphase figures.     
GenotypeHigh cyclin B at prometaphaseHigh cyclin B at metaphaseHigh cyclin B at anaphaseTotal mitotic cells scored
Wildtype (Canton S) 89% (59) 82% (23) 5% (18) 100 
idaΔ107/idaΔ107 96% (95) NA 84% (50) 145 
Parenthetical values are the number of cells scored for each stage. NA, ida cells do not contain metaphase figures.     

Discussion

IDA is a Drosophila homolog of APC5

We have presented a molecular and phenotypic analysis of ida, a putative homolog of APC5. Animals null for the ida locus fail to undergo metamorphosis, stall in early prepupal development, and die 2-3 days later. Examination of ida third-instar larvae reveals that they are lacking normally developed excorporate and incorporate imaginal discs because diploid primordial cells fail to proliferate during larval development. We also present evidence demonstrating that IDA is involved in germline cell proliferation, and a maternal component of ida+ is needed for egg production.

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 idamutants. 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 fzyprohibit 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.

Acknowledgements

The authors thank Carl Thummel for providing the λZAPII embryonic cDNA library. We also thank Bob Holmgren and Herman Dierick for critical comments on the manuscript. The monoclonal cyclin B antibody was obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa. This work was supported by NIH grants awarded to A.J.A. (GM54225) and M.L.G.(GM48430), and a Molecular Basis of Disease NIH Training Grant awarded to A.M.B.

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