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])

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 causes different pupal lethality. Cytological examination a key regulator of segregation and mitotic exit showed that these animals had an elevated level of in eukaryotes. It contains at least 11 subunits, most of apoptosis, high mitotic index and delayed or blocked which are evolutionarily conserved. The most abundant mitosis in a prometaphase-metaphase-like state with constituents of the vertebrate APC/C are the four overcondensed . The arrested neuroblasts structurally related tetratrico-peptide repeat (TPR) contained elevated levels of but, surprisingly, subunits, the functions of which are not yet precisely cyclin A appeared to be degraded normally. Contrary to understood. Orthologues of three of the TPR subunits have the situation for the Apc6/Cdc16 and Apc8/Cdc23 , the been identified in Drosophila. We have shown previously apparent loss of Apc7 function does not lead to the above that one of the TPR subunits of the Drosophila APC/C, abnormalities. Instead, the Apc7 knocked down animals Apc3 (also known as Cdc27 or Mákos), is essential for and null mutants are viable and fertile, although they development, and perturbation of its function results in display mild chromosome segregation defects and mitotic cyclin accumulation and metaphase-like arrest. In anaphase delay. Nevertheless, the Apc7 subunit shows this study we demonstrate that the Drosophila APC/C synergistic genetic interaction with Apc8/Cdc23 that, associates with a new TPR , a genuine orthologue of together with the phenotypic data, assumes a limited the vertebrate Apc7 subunit that is not found in yeasts. In functional role for Apc7. Taken together, these data suggest addition to this, transgenic flies knocked down for three of that the structurally related TPR subunits contribute the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by differently to the function of the anaphase-promoting

Journal of Cell Science RNA interference were established to investigate their complex. function. Whole-body expression of subunit-specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Key words: APC/C, TPR subunits, Apc6 (Cdc16), Apc7, Apc8 Apc8/Cdc23 silencing induces developmental delay and (Cdc23), Transgenic RNAi

Introduction identified in higher eukaryotes as well, indicating that the The -dependent protein degradation system controls structure of APC/C is evolutionarily conserved. Only the Apc9 mitotic progression by sequential degradation of different subunit appears to be specific to unicellular yeasts, whereas the regulatory . In this process a pathway of ubiquitin Apc7 subunit has so far been identified only in vertebrates activating (E1), ubiquitin conjugating (E2) and ubiquitin ligase (Harper et al., 2002). (E3) enzymes catalyse the conjugation of polyubiquitin chains Despite the essential role of APC/C in regulating mitosis, little onto substrate proteins, leading to their recognition and is known about the function of most of its individual subunits. degradation by the 26S proteasome. The key component of this The presence of structurally related proteins to the cullin proteolytic system is a multi-subunit ubiquitin ligase, the homologue Apc2, the RING finger Apc11 and the DOC1 anaphase-promoting complex (APC/C) that provides a domain containing Apc10 subunits in other E3 ubiquitin ligases platform and specificity for the ubiquitination reactions. The suggests direct roles of these subunits in the ubiquitination APC/C is active during mitosis and G1 phases of the reaction. Indeed, it was shown that the Apc2 and Apc11 subunits in which, by mediating securin and mitotic cyclin degradation, interact with each other (Ohta et al., 1999) and depending on the it triggers the metaphase to anaphase transition and the exit E2 enzyme, Apc11 alone or together with Apc2 could support from mitosis (Harper et al., 2002). the nonspecific transfer of ubiquitin to protein substrates The APC/C has been purified and analysed biochemically (Gmachl et al., 2000; Leverson et al., 2000; Tang et al., 2001) from yeast, Xenopus and clam egg extracts and from human in vitro. Furthermore, Apc10 was suggested as the processivity cells. It turned out to be a large protein complex containing at factor for the APC/C (Carroll and Morgan, 2002). least 13 stably associated core subunits in yeasts (Passmore, Much less is known about the function of other APC/C 2004). Orthologues of ten of the yeast subunits could be subunits, such as Apc1, Apc4 and Apc5. The ida coding TPR subunits of the APC/C have different functions 3239

for the Drosophila homologue of Apc5 has been cloned and X chromosome. EST sequences (LD05103 and LD39177) characterised. Mutant alleles of ida show a characteristic corresponding to this gene encode a 595 amino acid TPR- mitotic phenotype suggesting that this subunit controls some containing protein of approximately 68 kDa. Extensive sub-functions of the APC/C (Bentley et al., 2002). sequence comparisons, using BLAST, gapped BLAST and The Apc3 (also known as Cdc27 or Mks; hereafter referred PSI-BLAST programs (Altschul et al., 1997), showed 30% to as Apc3/Cdc27/mks), Apc6 (also known as Cdc16; referred identity and 51% similarity of the predicted protein along its to as Apc6/Cdc16), and the Apc8 (also known as Cdc23; referred entire length to the Apc7 subunit of the human APC/C (Yu et to as Apc8/Cdc23) subunits constitute a group of structurally al., 1998). Interestingly, predicted proteins from Arabidopsis related proteins within the APC/C, all of which contain nine to (accession no. NP 850309) and Apis mellifera (accession no. ten copies of the tetratrico-peptide repeat (TPR) motifs in XP 396165) came up with very similar topology to both the tandem arrays. The TPRs are repeats of 34 amino acid structural human and Drosophila proteins. In each of these proteins, the motifs with a consensus sequence restricted only to eight number, location and the distribution of the TPR motifs were residues. There is no invariant residue even within the consensus very similar: they all contained eight TPR motifs in a tandem but amino acids at these positions are conserved in terms of size, array at their C terminus and two single repeats toward their hydrophobicity and spacing (Lamb et al., 1995; Blatch and N-terminal end (Fig. 1A). Comparison of the ten TPR motifs Lässle, 1999). The first X-ray structure of the TPR containing of the CG14444 gene product to each other showed similarity protein phosphatase-5 revealed that each motif forms two ␣- mostly confined to the consensus sequence of this motif (Fig. helices in an antiparallel, helix-turn-helix configuration (Das et 1B). However, sequence comparison of individual motifs to al., 1998). The neighbouring motifs are packed in a parallel positionally equivalent TPR motifs of related proteins from fashion resulting in the formation of a superhelical structure. other species revealed that the conservation among these TPR motifs are present in functionally divergent proteins and repeats extends outside of the consensus (Fig. 1C), suggesting thought to mediate protein-protein interactions and the assembly functional correspondence. The similarity pattern outside the of multiprotein complexes. It is believed that the TPR subunits consensus is specific to individual TPRs. Thus these of the APC/C form a scaffold-like structure that facilitates the observations suggest that the CG14444 gene encodes a putative binding of other catalytic subunits, regulatory proteins and Apc7 protein in Drosophila melanogaster. Likewise, Apc7 substrates (Passmore et al., 2005). TPR subunits are essential for orthologues appear to exist in Apis mellifera and Arabidopsis viability in yeasts, and their mutant alleles result in uniform, thaliana as well, suggesting that this protein is present in all mitotic G2-M arrest phenotype (Lamb et al., 1994). multicellular eukaryotes. Owing to the high degree of In Drosophila, the genes coding for three TPR subunits, structural and sequential correspondence between CG14444 Apc3/Cdc27/mks, Apc6/Cdc16 and Apc8/Cdc23 have previously and the genuine vertebrate Apc7, we designate the Drosophila been identified (Deak et al., 2003; Harper et al., 2002; Huang gene as Apc7 in the rest of this paper. and Raff, 2002). Characterisation of Apc3/Cdc27/mks mutants indicated that this subunit is essential for APC/C function and Apc6/Cdc16- and Apc8/Cdc23-specific dsRNA required for the degradation of both cyclin A and cyclin B (Deak expression causes larval-pupal polyphasic lethality

Journal of Cell Science et al., 2003). In another study, GFP-tagged Apc3/Cdc27/Mks The inducible transgenic RNAi approach summarized in Fig. and Apc6/Cdc16 subunits localised differentially in living 2A was applied to knock down the expression of the syncytial embryos. Individual depletion of these subunits in Apc6/Cdc16, Apc7 and Apc8/Cdc23 genes. Constructs were cultured Drosophila S2 cells by RNA interference resulted in made in the pWIZ vector (Lee and Carthew, 2003) in which morphologically distinct mitotic phenotypes. These findings inverted doublets of cDNA sequences were placed under the suggest the existence of multiple forms of the APC/C with control of the GAL4-UAS system, and transgenic lines were different functions (Huang and Raff, 2002). established after transformation of isogenic flies. Expression To better understand the role of the TPR subunits in from these constructs leads to the synthesis of RNAs that are maintaining the structure and function of APC/C, we have predicted to snap back after splicing, and form loopless initiated a reverse genetic analysis on their homologues in dsRNAs. Several independent transgenic lines carrying single Drosophila melanogaster. First, the putative Drosophila copy insertions were isolated for each construct and mapped orthologue of the human gene Apc7 was identified through to the major chromosomes. All homozygous viable lines extensive sequence analysis, and then found to associate with showed comparable results. the APC/C complex. Following that, transgenic lines were To induce expression of transgenes, the RNAi strains were constructed carrying inducible RNA interference (RNAi) crossed to strains carrying the Act5C-GAL4 driver with a constructs specific for the Apc6/Cdc16, Apc7 and Apc8/Cdc23 ubiquitous expression of GAL4 throughout development (The genes. We show that knocking down these genes results in FlyBase Consortium, 2003). RT-PCR was used to determine strikingly different phenotypes that cover viability, cell cycle the expression of endogenous TPR subunit-specific mRNAs. progression, substrate degradation and apoptosis. These As shown on Fig. 2B, induction of all three dsRNA syntheses findings suggest that the TPR subunits contribute differentially was able to reduce TPR-specific mRNA levels by ~90% in to different sub-functions of the APC/C. early pupae relative to uninduced controls when normalized to the rpL17A calibration control. The da-GAL4 driver was also Results used for RNAi induction, and provided similar silencing effects The CG14444 gene encodes the Drosophila homologue to Act5C-GAL4 (data not shown). of Apc7 To examine how silencing of the TPR subunits affects The Drosophila genome project (BDGP) identified a gene viability, defined numbers of first instar larvae carrying single designated as CG14444 and localized to the 6C1 region of the copies of Act5C-GAL4 and either UAS-Apc6/Cdc16RNAi or 3240 Journal of Cell Science 120 (18)

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/Cdc23- specific 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 Fig. 1. Distribution and sequence comparisons of TPR motifs in the Apc3/Cdc27/mks gene. The mks1 mutation causes putative Apc7 orthologues. (A) Distribution of TPRs in the human Apc7 subunit (Hs), the Drosophila CG14444 gene product (Dm), and comparable reduction (~90%) of compared with the RNAi knock down effect in Apc6/Cdc16RNAi and predicted proteins from Arabidopsis thaliana (At; accession no. NP RNAi 850309) and Apis mellifera (Am; accession no. XP 396165). The Apc8/Cdc23 animals, yet these mutants die as late pharate asymmetric rectangular symbols represent single TPR motifs. adults with severe rough eye and bristle phenotypes (Deak et (B) Sequence alignment of seven TPR motifs of the Drosophila al., 2003). Apc7 protein. The TPR consensus sequence is shown below the alignment. Residues matching the consensus are in bold type and Apc6/Cdc16- and Apc8/Cdc23-specific gene silencing highlighted in grey (also in C). Residues are conserved only at the results in metaphase-like arrest with overcondensed consensus positions but, even there, there is no invariant position. chromosomes (C) Sequence comparison of the TPR10 motif from the proteins To determine whether Apc6/Cdc16 or Apc8/Cdc23 silencing shown at the top of this figure. Conserved residues outside the consensus are highlighted in yellow. Residue conservation is more affects cell cycle progression, we examined orcein-stained extensive both within and outside of the consensus. 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 RNAi

Journal of Cell Science UAS-Apc8/Cdc23 constructs were collected and their either scattered all over the cells or congregated at the survival was monitored by counting the number of third instar metaphase plate (Fig. 3E-G,I-K). The chromosome larvae and pupae. Induction of Apc6/Cdc16- and Apc8/Cdc23- overcondensation indicated that the cells were delayed or specific RNAi did not significantly affect survival of embryos arrested in mitosis, as chromosomes continue condensation and early larvae. However, lethality was observed in the last during this time. Accordingly, the proportion of cells in mitosis larval (L3) stage, and all animals died as pupae. In addition to was significantly higher, and this leads to about double the this, the third instar larvae showed a 1- to 2-day delay in mitotic index (MI) of the wild type (Table 2). Most of the puparium formation and frequently these larvae formed mitotic cells were in a prometaphase-metaphase-like state, and melanotic tumours (Table 1A). No melanotic tumours or 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 3241

Table 1. Lethal phase of TPR genes knocked down by RNA interference Genotype L3 Larvae* (%) Prepupal stage P3 (%) Prepupal stage P4(i) (%) Prepupal stage P4(ii) (%) Pupal stage P5(i) (%) A Apc6RNAi 6 + mt 3 – 11 86 Apc7RNAi Viable Viable Viable Viable Viable Apc71 Viable Viable Viable Viable Viable Apc8RNAi 13 + mt 20 – 65 15 B Apc6RNA-Apc7RNAi 7 + mt 3 – 15 82 Apc6RNAi-Apc8RNAi 27 + MT 26 70 4 – Apc7RNAi-Apc8RNAi 20 + MT 28 70 2 –

*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 three- RNAi induced mitotic cells the chromosomes appeared to times higher metaphase-anaphase ratios (M:A) in the congregate precisely to the metaphase plate (Table 2 and Fig. Apc6/Cdc16 and Apc8/Cdc23 RNAi preparations, respectively 3F,J). This true metaphase stage was quite rare or indeed could (Table 2), and suggests that loss of function of these subunits not be detected in Apc3/Cdc27/mks, Apc5/ida and Apc2/mr leads to metaphase-like delay or arrest. mutants, and was significantly higher than that seen in the wild The scattered distribution of chromosomes in RNAi induced type (2-3%). The chromosomes at the metaphase plate were Apc6/Cdc16 and Apc8/Cdc23 cells were very similar to the also overcondensed, indicating that the delay or arrest was phenotype caused by the Apc3/Cdc27/mks, Apc5/ida and maintained during that stage as well. Apc2/mr mutations (Deak et al., 2003; Bentley et al., 2002; In some of the arrested cells the chromosomes appeared to Reed and Orr-Weaver, 1997). undergo decondensation before attempting to complete However, in about 10% of Apc6/Cdc16 and Apc8/Cdc23 anaphase and cytokinesis (Fig. 3G). It has been suggested that Journal of Cell Science

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). 3242 Journal of Cell Science 120 (18)

Table 2. Mitotic phenotypes of TPR genes knocked down by RNA interference Genotype Preparations/fields* Mitotic index (MI)† Metaphase:anaphase ratio Chromosomes at metaphase plate (%)‡ Polyploid (%)‡ A Canton S 4/60 1.7±0.3 1.9±0.3 2.7 0 Apc6RNAi 4/60 3.2±0.3 4.2±0.5 11.0 4.6±1.8 Apc7RNAi 6/70 2.1±0.3 1.4±0.4 2.2 0 Apc8RNAi 4/50 3.5±0.4 5.2±0.3 9.7 8.1±0.9 B Apc6RNAi-Apc7RNAi 5/90 3.5±0.3 5.5±0.5 10.0 3.0±0.8 Apc6RNAi-Apc8RNAi 5/50 4.4±0.4 6.6±0.8 11.6 7.7±3.0 Apc7RNAi-Apc8RNAi 4/40 4.2±0.4 6.3±0.5 9.3 6.2±2.1

*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 have lesser roles in cyclin A removal but are required for cyclin chromosomes (Gatti and Baker, 1989). Following DNA B degradation. replication, such cells become tetraploid, or with the recurrence of events, polyploid. A proportion of Apc6/Cdc16 and Apc7 is a nonessential protein that associates with the Apc8/Cdc23 RNAi cells were polyploid, most frequently with Drosophila APC/C highly overcondensed tetraploid or less frequently octaploid or As Fig. 2B demonstrates, RNA interference caused significant higher ploidy chromosome complements (Table 2 and Fig. reduction in the expression of the Apc7 gene. This effect was 3G,K). confirmed independently by using quantitative fluorescent In addition to metaphase, irregular chromosome behaviour real-time PCR that showed greatly delayed amplification in could be detected in anaphase as well, which included lagging Apc7RNAi samples compared with controls (data not shown). chromosomes and chromosome bridges (Fig. 3L). The delayed amplification is consistent with lower concentration of Apc7 transcripts in those samples. Although Apc6/Cdc16 and Apc8/Cdc23 RNAi lines arrest with the degree of gene silencing was at least as effective as in the high levels of cyclin B but with apparent destruction of Apc6/Cdc16 and Apc8/Cdc23 RNAi lines, the Apc7RNAi cyclin A animals developed without any delay or abnormalities and One of the main functions of APC/C is to aid the degradation hatched as fertile adults (Table 2). of mitotic cyclins. Previously, we have shown that mutations Orcein-stained brain squash preparations from Apc7RNAi (mks1 and mksL7123)in the Apc3/Cdc27/mks gene stabilized third instar larvae lack any sign of prometaphase or metaphase- both cyclin A and B in the mitotically arrested cells (Deak et like arrest characteristic of the other TPR subunit mutants. al., 2003) (M.P. and P.D., unpublished). We were interested to Instead, chromosomes appeared somewhat more tangled and

Journal of Cell Science follow mitotic cyclin levels in RNAi induced neuroblasts by exhibited signs of uneven condensation together with frequent immunostaining with polyclonal antibodies raised against chromosome bridges in anaphase (Fig. 3D,H). The number of cyclin A and cyclin B. In wild-type cells, cyclin A could be cells in mitosis remained low but the proportion of the detected in prophase and early prometaphase (Fig. 4A-D), but anaphase and telophase figures approximated the number of it became undetectable around metaphase and early anaphase cells in prometaphase and metaphase, resulting in a mitotic (Fig. 4E-H). Cyclin B degradation follows that of cyclin A, so index that was similar to that of wild type, and a metaphase- its level declines rapidly in metaphase, and at and after anaphase ratio (M:A) that was lower than in wild type (Table chromosome segregation (Fig. 5A-H). As expected, both cyclin 2). The lower M:A ratio may indicate some delay during A- and cyclin B-specific antisera gave strong staining in the anaphase in the Apc7RNAi cells. RNAi induced Apc6/Cdc16 and Apc8/Cdc23 preparations. In order to further support the nonessential nature of the Closer inspection revealed that the cells strongly stained by Apc7 gene, a null mutant allele (Apc71) was isolated by cyclin A-specific antibody gave rather weak and somewhat imprecise excision of the P{EPgy2}EY11333 element diffuse DNA signal, indicative of being in prophase or early localized 63 bp distally from the 3Ј end of the gene. The prometaphase. The majority of cells (80%) arrested in late remobilization generated a 1576 bp deletion extending from prometaphase or in metaphase were not stained by anti-cyclin the insertion site toward the 5Ј end of Apc7. This internal A antibody above background level (Fig. 4I-L,M-P). However, deletion removed about two-thirds of the Apc7 gene, including more than 80% of cells that stained intensely with cyclin B- the functionally important eight tandem TPR repeats specific antibody also showed bright staining of DNA at the (Vodermaier et al., 2003). The Apc71 homozygotes appeared metaphase plate and a spindle indicative of being in metaphase viable and fertile, and could be maintained as a homozygous arrest with overcondensed or polyploid sets of chromosomes stock. Their mitotic phenotype was indistinguishable from that (Fig. 5I-L,M-P). Thus, it appears that in terms of cyclin A of Apc7RNAi lines. To determine the role of Apc7 in cyclin degradation, the Apc6/Cdc16 and Apc8/Cdc23 mitotic degradation, we monitored cyclin levels in Apc7RNAi and Apc71 phenotypes differ from that of Apc3/Cdc27/mks mutants, neuroblast preparations by immunostaining. We could not where both cyclin A and cyclin B accumulate (Deak et al., detect abnormal changes in the level and localisation of either 2003). The apparent degradation of cyclin A and the cyclin A or cyclin B in mitotic cells and their turnover appeared accumulation of cyclin B in cells with diminished Apc6/Cdc16 similar to that in wild type (data not shown). and Apc8/Cdc23 functions indicate that these subunits may To see whether Apc7 is a genuine component of the TPR subunits of the APC/C have different functions 3243

Drosophila APC/C, we examined physical interaction of total protein extracts (lanes 1 and 2). These data show that between Apc7 and universal APC/C subunits Apc8/Cdc23 or at least a fraction of Apc7 is clearly associated with the APC/C Apc3/Mks by co-transfection of S2 cells and affinity in Drosophila, but perhaps not as a core subunit. Possible chromatography. FLAG-tagged Apc7 (F7) was co-expressed interpretations of the low-yield co-purification of Apc7 in the in S2 cells with either haemagglutinin (HA) epitope-tagged FLAG-Apc8 pull-down complex is that Apc7 either interacts Apc8/Cdc23 (H8) or Apc3/Mks (H3), and then FLAG-Apc7 only with certain forms of the APC/C complex or it interacts and its associated proteins were pulled down from cell extracts only transiently with the APC/C. with an anti-FLAG affinity column. Following repeated washing, bound proteins were eluted with an excess of free Apc8/Cdc23RNAi show synergism with Apc6/Cdc16RNAi FLAG peptide, and detected by western analysis using and Apc7RNAi monoclonal anti-FLAG or anti-HA antibodies. Fig. 6 shows TPR subunits are thought to be involved in protein-protein that both HA-Apc3/Mks (lanes 7 and 8) and HA-Apc8 (lanes interactions to form large protein complexes (Blatch and 3 and 4) were retained on the column with FLAG-Apc7 under Lässle, 1999), and were suggested to interact with each other washing condition of 0.15 M NaCl (in 20 mM Tris), but there within the budding yeast APC/C (Lamb et al., 1994). Since was no detectable retention without Apc7 (Fig. 6, lanes 9 and genetic interaction tests of double mutants are powerful 10). However, in a reciprocal experiment, when FLAG-Apc8 techniques to investigate functional relationships either was co-expressed with HA-Apc7, and FLAG-Apc8-associated between genes or their protein products, we applied this to our proteins were pulled down, only a faint band of HA-Apc7 RNAi lines. Transgenic strains carrying two RNAi constructs could be detected (Fig. 6, lane 6), indicating a weak in different combinations were generated and crossed to association of HA-Apc7 with FLAG-Apc8. The expression Act5C-GAL4 partners to induce gene silencing. Again, defined levels of the different N-terminal fusion constructs were number of first instar larvae carrying single copies of Act5C- comparable in these experiments as judged from western blots GAL4 and pair-wise combinations of the UAS- Journal of Cell Science

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 wild- type 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)

Apc6/Cdc16RNAi, UAS-Apc7RNAi or UAS-Apc8/Cdc23RNAi pronounced mitotic arrests phenotype of the double RNAi lines constructs were collected and their lethal phase and mitotic suggest a firm synergistic interaction between Apc8/Cdc23 and phenotypes were determined. Apc6/Cdc16 as well as Apc8/Cdc23 and Apc7, thus implicating Similarly to single RNAi lines, the embryonic and early that these subunits contribute independently to the same larval developments were not affected in any of the double function of the APC/C. RNAi combinations. However, as Table 1 illustrates, the late The phenotype of the third double RNAi line, larval lethality and the number of larvae with melanotic Apc6/Cdc16RNAi-Apc7RNAi appeared different from the ones tumours were greatly increased in the Apc6/Cdc16RNAi- discussed above. The lethal phase and mitotic phenotype were Apc8/Cdc23RNAi (27%) and Apc7RNAi-Apc8/Cdc23RNAi (20%) similar to those of Apc6/Cdc16RNAi alone (Table 1), the combinations compared to Apc6/Cdc16RNAi (6%), Apc7RNAi hallmark of epistatic interaction. However, as one of the lines, (viable) or Apc8/Cdc23RNAi (13%) alone. The Apc6/Cdc16RNAi- Apc7RNAi, is viable with mild mitotic phenotype, one would Apc8/Cdc23RNAi and the Apc7RNAi-Apc8/Cdc23RNAi double also expect the double mutant phenotype to reflect the stronger mutants also showed a definite shift in pupal lethality toward mutant if these gene products act independently, therefore no earlier stages compared to the single RNAi lines (Table 1). obvious interaction can be established between Apc6/Cdc16 The range of mitotic phenotypes found in the and Apc7. Apc6/Cdc16RNAi-Apc8/Cdc23RNAi and the Apc7RNAi- Apc8/Cdc23RNAi double RNAi lines was essentially the same Apc6/Cdc16- and Apc8/Cdc23-specific RNA interference as that found in the Apc6/Cdc16RNAi and Apc8/Cdc23RNAi lines induces apoptosis alone; however, the MI of these double RNAi combinations In addition to mitotic cells, we observed many pycnotic nuclei were significantly higher (4.4 and 4.2, respectively; P<0.05) in the orcein-stained preparations (Fig. 7A,B). The cells were than that of Apc6/Cdc16RNAi (3.2), Apc7RNAi (2.1) or rounded, relatively small and their nuclei showed strong but Apc8/Cdc23RNAi (3.5) alone (Table 2). Taken together, the uneven staining. Pycnotic nuclei characteristic of apoptotic increased L3 lethality, the shift of lethal phase and the more cells could be found sporadically in wild-type tissues as well, Journal of Cell Science

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 wild- type 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

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.

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 Fig. 6. FLAG affinity chromatography of FLAG-tagged Apc7 and involvement in the formation of substrate-ubiquitin conjugates, Apc8 complexes. (A) Western blots of total protein extracts from S2 and thus determines substrate specificity of the whole process. cells transfected with HA-tagged Apc8 (H8, lane 1) or FLAG-tagged More than half of the total mass of the APC/C consists of Apc7 (F7, lane 2) plasmids. Strip 1 was reacted with anti-HA (␣H, tandem arrays of TPR motifs present in several evolutionarily lane 1), and strip 2 with anti-FLAG (␣F, lane 2) monoclonal conserved subunits. Little is known about the specific functions antibodies. (B) Western blots of proteins affinity-purified on anti- of the TPR subunits, although a structural role to form a FLAG-M2 antibody beads from S2 cells co-transfected with FLAG- scaffold-like core of the APC/C has been suggested (Passmore Apc7 and HA-Apc8 (F7-H8, lanes 3 and 4), FLAG-Apc8 and HA- et al., 2005). The TPR subunits in budding yeast are coded by Apc7 (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, the Cdc27, Cdc16 and Cdc23 genes that are all essential with 5 and 7) or anti-HA (lanes 4, 6 and 8) monoclonal antibodies. The uniform loss-of-function mitotic arrest phenotypes. We show immunoreactive bands labelled with an asterisk are proteolytic here that in addition to the orthologues of these subunits, a gene degradation products of Apc8. (C) Blots of anti-FLAG-M2 column- is present in the Drosophila genome that codes for a protein bound (lane 10) and unbound (flow-through, lane 9) proteins from closely related to the Apc7 subunit of the vertebrate APC/C. S2 cells transfected with HA-Apc8 (H8) plasmid and incubated with Our data support the nomination of this protein as the anti-HA monoclonal antibody. Even after heavily overloading the Drosophila Apc7 homologue. Firstly, its primary sequence anti-FLAG column, no nonspecific binding of HA-Apc8 could be appears to be conserved, including the number, location and detected. Similarly, HA-Apc3 did not bind to the anti-FLAG-M2 distribution of its TPR motifs. The TPR motifs in the column (data not shown) E, eluate; FT, flow-through. 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. but their frequency increased by three- to fourfold in Secondly, the putative Apc7 protein is associated with

Journal of Cell Science Apc6/Cdc16 (P<0.005) and five to sixfold in Apc8/Cdc23 universal subunits of the Drosophila APC/C. Our affinity (P<0.005) knocked down preparations (Fig. 7E), suggesting purification data suggest that this association is either weak, or strong induction of apoptosis. To confirm that cell death was occurs only in certain forms of the APC/C complex. The induced in these RNAi lines, we stained larval brains with existence of multiple forms of the APC/C has previously been Acridine Orange. As Fig. 7C,D illustrates, the Acridine Orange 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. 3246 Journal of Cell Science 120 (18)

reported in budding yeast (Passmore et al., 2005). with the delay in chromosome alignment reported for the Alternatively, it is also possible that Apc7 is not a core subunit overexpression of cyclin A in HeLa and PtK1 cells (den Elzen but a transiently interacting nonessential partner of the and Pines, 2001) and overexpression of stable cyclin AS in Drosophila APC/C. Drosophila embryos (Parry et al., 2003). In Apc6/Cdc16RNAi Thirdly, the putative Drosophila Apc7 gene shows and Apc8/Cdc23RNAi cells, degradation of cyclin A facilitates synergistic genetic interaction with the Apc8/Cdc23 gene that chromosome alignment, whereas the imposed mitotic arrest also implicates a functional relationship. Although the idea was prolongs metaphase and hence the higher number of cells with that Apc7 is only a vertebrate-specific subunit of the APC/C overcondensed but precisely aligned chromosomes. The (Yu et al., 1998), it is now shown to be also present in species chromosomes of cells escaping from or passing this phase from plants and insects, suggesting that this protein became become scattered similarly to mks1 arrested cells. This associated with the APC/C in most multicellular organisms. appearance is the result of uncoordinated, oscillating poleward We have previously shown that one of the TPR genes in movements of chromosomes caused by the accumulation of Drosophila, Apc3/Cdc27/mks, is essential and involved in both cyclin B (Parry and O’Farrell, 2001). Since a high level of cyclin A and cyclin B degradation (Deak et al., 2003). To cyclin A has a similar effect on chromosome movements in the address the function of the other TPR proteins, we established arrested cells as cyclin B (den Elzen and Pines, 2001), the transgenic RNAi lines to reduce Apc6/Cdc16-, Apc7- and accumulation of both cyclin A and B prevents chromosome Apc8/Cdc23-specific gene expression in intact animals. alignment in mks1 cells. Knocking down the expression of Apc6/Cdc16 or Apc8/Cdc23 The diverse phenotypic features of the TPR mutants are genes caused lethality, indicating that as in yeast, these somewhat surprising and appear to be inconsistent with the genes are essential in Drosophila as well. Furthermore, predicted role of TPR subunits to form a central scaffold-like Apc6/Cdc16RNAi and Apc8/Cdc23RNAi cells show mitotic structure to mediate APC/C assembly (Vodermaier et al., 2003; phenotypes with high mitotic index, overcondensed Passmore et al., 2005). One would expect such a scaffold to chromosomes and metaphase-like arrest that is very similar to become unstable or collapse when its components are removed the mr, mks1 and ida mutants that code for the Apc2, one by one or in double mutant combinations, leading to Apc3/Cdc27/Mks and Apc5 subunits of the Drosophila inactive APC/C, but we could not detect that in our RNAi lines. APC/C, respectively (Reed and Orr-Weaver, 1997; Deak et al., Instead, all single and double TPRRNAi lines had slightly 2003; Bentley et al., 2002). This hallmark phenotype supports smaller than normal larval brains and imaginal discs that the assumption that the Apc6/Cdc16 and Apc8/Cdc23 genes indicate reasonable mitotic activity and the existence of an code for genuine, functionally conserved APC/C subunits in APC/C with at least residual functions. Since the TPR subunits Drosophila. show extensive structural similarities, it is conceivable that a Unlike in yeast, however, the phenotypes of the TPR mutants missing subunit can be substituted by another one. This could are not uniform, since beyond common characteristics, they theoretically explain the relative stability of the scaffold show significant differences. Whereas the Apc7RNAi and Apc71 structure but it would not account for the different phenotypes individuals are viable, mks1 mutants die as pharate adults, of the TPR subunits. Therefore, the data presented here better RNAi

Journal of Cell Science Apc6/Cdc16 animals die as P5(i) early pupae, and fit an alternative model of APC/C that is centred on the largest Apc8/Cdc23RNAi animals die as P4(ii) prepupae. Differences subunit, the Apc1/Shtd (Shattered in Drosophila), as a scaffold can also be seen in the ability to degrade APC/C substrates, (Thornton et al., 2006). In this model, the Apc1/Shtd provides such as the mitotic cyclins. In this regard, removal of Apc7 has a platform to which all the other subunits bind by forming no effect, depletion of Apc6/Cdc16 and Apc8/Cdc23 appear to functional subcomplexes. Consistently with this model, the affect mainly cyclin B, whereas loss of Apc3/Cdc27/Mks loss-of-function Apc1/shtd Drosophila mutants express an affects both cyclin A and B degradation. These observations extremely strong mitotic phenotype including larval lethality, suggest different requirements of the TPR subunits in mitotic lack of imaginal discs, strong metaphase arrest with no or rare cyclin degradation. However, it should be noted that these anaphases, and very high frequency of polyploidy (M.P. and differences could also be due to different inactivation P.D., unpublished). The mitotic phenotype of Apc1/shtd efficiencies, despite the fact that the residual expression levels mutants appears stronger than all the other known APC/C of the genes appear to be very similar in all TPR mutants as subunit mutants, and RNAi lines presented here, suggesting judged by RT-PCR (Fig. 2B) (Deak et al., 2003). In this case complete loss of APC/C structure and function. Taken all data depletion of individual TPR subunits may affect the affinity of together, our view is that the TPR subunits may form functional substrate binding, or the efficiency of the ubiquitylation subcomplexes of the APC/C that could bind to the Apc1/Shtd reaction leading to an APC/C activity level at which cyclin A scaffold and most probably be involved in activator and degradation is still sufficient, whereas cyclin B degradation is substrate binding. not. Removal of most of the TPR subunits seems to have a direct The metaphase-like arrest of Apc6/Cdc16RNAi and effect on cell viability as well. This is apparent from the Apc8/Cdc23RNAi cells is unique in that they frequently show de significant increase in the number of apoptotic cells in the facto metaphase arrest with overcondensed chromosomes mitotically active larval brains of Apc6/Cdc16RNAi and aligned precisely at the metaphase plate. This correlates with Apc8/Cdc23RNAi (Fig. 7), or mks1 animals (Deak et al., 2003). the ability of these cells to degrade cyclin A. This is in stark These perturbations of APC/C function cause stabilization of contrast with mks1 cells that accumulate cyclin A and do not, cyclin B, thus leading to prolonged activation of the mitotic or seldom, form literal metaphase figures. These observations kinase, Cdc2, at the metaphase to anaphase progression and suggest that cyclin A degradation is required in Drosophila for beyond, when its activity must normally plummet to allow proper chromosome alignment at metaphase. This is consistent mitosis to proceed. A plausible explanation for the high level TPR subunits of the APC/C have different functions 3247

of apoptosis is that the extended active state of Cdc2 during stages of mitosis was determined for each single or double mutant line and compared using Student’s t-test. late mitosis and mitotic exit could induce cell death. High Cdc2 Quantification of cell death was performed by observing the morphology of activity has been found in apoptotic conditions and shown to orcein-stained cells, and scoring for apoptotic cells with pycnotic nuclei. The mean be required for the induction of this process (Fotedar et al., number of apoptotic cells was determined and used for further statistical analysis. 1995; Konishi et al., 2002). Since the molecular details of Cdc2 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 action are not fully known, further investigation is required to (C.I. 46005, Molar Chemicals) solution and incubated for 5 minutes in the dark, link cell cycle regulation to the cell death machinery. 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 Materials and Methods preparations were examined using an Olympus FV1000 confocal microscope within Fly stocks 15-20 minutes from dissection. 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 Epitope constructs and affinity chromatography described by Lindsley and Zimm (Lindsley and Zimm, 1992). Stocks were obtained Plasmid constructs to express N-terminal FLAG and haemagglutinin (HA) from the Bloomington Stock Center unless stated otherwise. For phenotypic epitope-tagged proteins in S2 cells under the control of the Actin-5C promoter analysis, the chromosomes carrying a transgenic construct were balanced over CyO, were constructed using the Drosophila Gateway Vector System ActGFP or T(2;3)TSTL, CyO: TM6B, Tb. 1 (http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html). First, full-length The Apc7 mutant was established after imprecise excision of the cDNAs of Apc3/Cdc27/mks, Apc7 and Apc8/Cdc23 were cloned into the appropriate P{EPgy2}EY11333 element localized 63 bp downstream of the predicted stop entry (pENTR) vectors in frame. Following that, the ORFs of these genes were codon of CG14444 (Bellen et al., 2004). Five lines were recovered carrying internal recombined either into the pAFW or pAHW destination vectors using Gateway LR deletions of this gene. The largest deletion removed 1576 bp, about two-thirds of clonase II Enzyme Mix (Invitrogen). The pAFW vector contains three FLAG Ј the gene, starting at the insertion site and extending toward the 5 end of the Apc7 whereas the pAHW vector contains three HA epitope tags 5Ј of the Gateway gene. The insertion site is marked by a 24 bp long remnant of the original cassette. Successful recombinant clones were selected based on their resistance to P{EPgy2}EY11333 element. The resulting truncated Apc7 protein lacks two-thirds ampicillin and lack of ccdB toxicity. All clones were verified by sequencing before of its mass including the eight tandem TPR repeats. This mutation was designated 1 transfection. The N-terminal fusion constructs were designated in this paper as as Apc7 and used in this work as a knocked out allele of the Apc7 gene. 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 pAHW- Transgenic RNA-interference lines Apc8 as HA-Apc8 or H8. To make transgenic double-stranded RNA-interference constructs, PCR primer pairs Schneider 2 (S2) tissue culture cells were either transfected with 2.5 ␮g plasmid of 5Ј-ACTCTAGATTGCCTGTCTGGTGGAAAAC-3Ј and 5Ј-ATTCTAGACT - DNA of FLAG-Apc7, HA-Apc7, HA-Apc3, FLAG-Apc8 or HA-Apc8 alone or co- TGCGCAGCGAATGACC-3Ј; 5Ј-ACTCTAGAGCTCTCGCCCAAATGTTCA-3Ј transfected with 2.5+2.5 ␮g FLAG-Apc7+HA-Apc3, FLAG-Apc7+HA-Apc8 or and 5Ј-ATTCTAGAGAGGCGTATCGTCCAGTTGC-3Ј; and 5Ј-ACTCTAGAAC - FLAG-Apc8+HA-Apc7 double combinations using 15 ␮l Cellfectin (Invitrogen) in GCGCCCTGAAACTGAAT-3Ј and 5Ј-ATTCTAGATCGACGCATGCCCTGAAT- serum-free S2 cell medium (Sigma). For preparation of total protein extracts, 3Ј were used to amplify 656 bp, 917 bp and 910 bp sequences corresponding to transfected cells were dissolved in SDS sample buffer 48 hours after transfection. exon 5, exon 3 and exon 2 and 3 of the Drosophila Cdc16, Apc7 and Cdc23 For affinity chromatography, cells were washed in PBS 48 hours after transfection transcripts, respectively (XbaI sites are underlined). The PCR products were cloned and cell extracts were prepared by incubating the cells in 25 mM Hepes pH 7.6, into the pWIZ vector (a gift from Richard Carthew, Northwestern University, USA), 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and 0.2% in opposite orientation on both sides of the white intron present in the vector (Lee NP40. After 10 minutes incubation on ice, an equal volume of cytoplasmic dilution and Carthew, 2003). The obtained gene-specific RNAi constructs were designated buffer (25 mM Hepes pH 7.6, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM as P{UST-Cdc16RNAi}, P{UST-Apc7RNAi} and P{UST-Cdc23RNAi}, and all EGTA, 1 mM PMSF and 450 mM NaCl) was added, the nuclei were removed by constructs were verified before being processed for transformation. Following P- centrifugation (3 minutes at 10,000 g) and FLAG-tagged proteins were affinity- element-mediated germline transformation (Spradling and Rubin, 1982) of w1118 purified on anti-FLAG-M2-agarose beads (Sigma-Aldrich). After 2 hours of Journal of Cell Science isogenic flies, at least five independent transgenic lines were obtained for each gene. incubation at 4°C, anti-FLAG-M2-agarose beads were washed four times with five To test RNAi effects, homozygous transgenic flies carrying subunit-specific RNAi volumes of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and bound proteins were eluted constructs (such as P{UST-Cdc16RNAi}) were crossed to Act5C-GAL4 females with an excess of FLAG peptides. Total protein extracts or affinity-purified samples which expressed the Gal4 transcription factor under the control of the actin5C were fractionated on 8% SDS-PAGE and blotted to PVDF membrane strips. Western promoter. The progeny of these crosses were examined for subunit-specific gene blots were reacted with anti-FLAG or anti-HA monoclonal antibodies. expression as well as lethal and mitotic phenotypes. Immunostaining RT-PCR Third instar larval brains (n=3-6) were dissected in PBS, fixed in ice-cold methanol Total RNA was isolated from young pupae using the Tri Reagent extraction kit for 20 minutes, washed three times for 5 minutes in PBS and than permeabilised in (Sigma). The RNA samples were DNase treated with RQ1 RNase-Free DNase PBS containing 0.3% Triton X-100 for 10 minutes at room temperature and washed (Promega). cDNA was synthesized from 5 ␮g total RNA as template with M-MuLV again in PBS containing 1% BSA for 40 minutes at room temperature. The brains reverse transcriptase (Fermentas, Vilnius, Lithuania) and random hexanucleotide were incubated with primary antibodies diluted to an appropriate concentration in primers (Fermentas, Vilnius, Lithuania). Two primers complementary to the 4. and PBS containing 1% BSA overnight in a humid chamber. They were than washed 5. exons of Apc6, to the 2. and 3. exons of Apc7 and to the 1. and 3. exons of Apc8 four times for 15 minutes each with PBST (PBS supplemented with 0.1% Triton were used in 20 cycle PCR amplifications. 20 cycle PCRs were performed using X-100) containing 1% BSA at room temperature before being incubated with rpL17A primers (rpL17A upper, 5Ј-GTGATGAACTGTGCCGACAA-3Ј; rpL17A appropriate secondary antibodies (Jackson) diluted in PBST containing 1% BSA lower, 5Ј-CCTTCATTTCGCCCTTGTTG-3Ј) on the same cDNA templates to serve for 2 hours at room temperature in the dark. The preparations were finally washed as calibration controls. RT-PCR products were separated by agarose gel twice for 15 minutes in PBST and two times 15 minutes in PBS. DNA was stained electrophoresis. with TOTO-3 (Molecular Probes) before the specimens were mounted in Vectashield. Cytology The primary antibodies were Rb270 rabbit anti-cyclin A antibody (Whitfield et Brains from third instar larvae were dissected in a large drop of 0.7% NaCl solution al., 1990), Rb271 rabbit anti-cyclin B antibody (Whitfield et al., 1990) and rat then transferred to a drop of 45% acetic acid on a microscope slide. After 10-15 monoclonal anti-␣-tubulin antibody, YL1/2 (Kilmartin at al., 1982). The seconds, the liquid was absorbed with a piece of filter paper and replaced with a preparations were examined using an Olympus FV1000 confocal microscope. For drop of 3% orcein (Gurr’s 23282) in 45% acetic acid. After 3-5 minutes staining, comparing cyclin levels, images of metaphase cells were acquired using identical the orcein was removed and the brains were destained in a drop of 65% acetic acid settings and cyclin immunostaining was determined by visual inspection of the for 5-10 seconds. Following that, the destaining solution was replaced with a drop images. 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 We wish to thank Katalin Udvardy for her contribution in carrying preparations were examined and photographed under phase-contrast optics using an out the S2 cell transfection experiments. We are grateful to Mary Lilly Olympus BX51 upright microscope with a UPLFLN 100XO2PH plan-apochromat objective. A DP 70 digital colour camera was used to take images. Mitotic index and Edward Wojcik for discussions and comments on the manuscript. (MI) was defined as the number of mitotic cells on an optical field. On average, 10- This work was supported by a Hungarian Scientific Research Fund 20 optical fields per preparation were scored. The mean number of cells at different (OTKA) grant awarded to P.D. (T037322). 3248 Journal of Cell Science 120 (18)

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