4208 Research Article Loss of the mammalian APC/C activator FZR1 shortens G1 and lengthens S phase but has little effect on exit from mitosis

Reinhard Sigl1, Cornelia Wandke1, Veronika Rauch1, Jane Kirk2, Tim Hunt2 and Stephan Geley1,* 1Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria 2Clare Hall Laboratories, Research UK, South Mimms, England, UK *Author for correspondence ([email protected])

Accepted 10 September 2009 Journal of Cell Science 122, 4208-4217 Published by The Company of Biologists 2009 doi:10.1242/jcs.054197

Summary The anaphase-promoting complex/cyclosome (APC/C) is knockdown of p53 in U2OS cells with inducible FZR1 siRNA essential for progression through mitosis. At anaphase onset, also failed to restore their proliferative capacity. Thus, the the APC/C requires the activator CDC20 to target proliferation defects are a direct consequence of the genetic securin and cyclin B1 for proteasome-dependent degradation, damage inflicted by loss of FZR1 function and are largely but then depends on the CDC20-related protein FZR1 (also independent of p53. In summary, mammalian FZR1 is not known as CDH1) to remain active until the onset of the next S required for the completion of mitosis, but is an important phase. To investigate the role of FZR1 in mammalian cells, we regulator of G1 phase and is required for efficient DNA used RNAi in human cell lines and conditional targeting replication in human and mouse somatic cells. in mouse embryonic fibroblasts. In neither case was FZR1 required for exit from mitosis, but in cells lacking FZR1, the Supplementary material available online at G1 phase was shortened and the S phase was prolonged. In http://jcs.biologists.org/cgi/content/full/122/22/4208/DC1 several normal and transformed human cell lines, loss of FZR1 function induced DNA-damage responses and impaired Key words: FZR1, APC/C, Mitosis, Proteolysis, Cyclin, , proliferation independently of the p53 status. Constitutive p53, Replication, DNA damage

Introduction al., 2005). In contrast to APC/C-CDC20, the FZR1-associated form

Journal of Cell Science The anaphase-promoting complex/cyclosome (APC/C) is a large of the APC/C has a different substrate spectrum. In addition to D- multisubunit ubiquitin protein ligase that is essential for sister box-containing , the APC/C-FZR1 also targets other proteins chromatid separation and exit from mitosis. Because these events with different degrons, such as the KEN box present in CDC20 are crucial for equational segregation, the activity of (Pfleger and Kirschner, 2000). The switch from CDC20- to FZR1- the APC/C is tightly controlled by several mechanisms, including activated APC/C occurs during anaphase B (Hagting et al., 2002) cyclin-dependent kinase (CDK)-dependent phosphorylation and the and it is believed that FZR1, by targeting Aurora kinase A and PLK1, binding of cell division control protein 20 (CDC20). Once activated, contributes significantly to APC/C-dependent proteolysis during exit the APC/C recognises its substrates via specific motifs, such as the from mitosis (Pines, 2006). destruction boxes (D-box) present in cyclin B1 and securin. Its After completion of mitosis, the APC/C remains active until the activity against these essential substrates is, however, constrained next S phase (Brandeis and Hunt, 1996) and several APC/C by the mitotic checkpoint to prevent premature sister chromatid substrates either remain or become unstable in G1 phase. Degradation separation and exit from mitosis, which can generate aneuploid of some of these substrates is important for maintenance of G1 phase progeny (reviewed by Peters, 2006). In contrast to the ubiquitylation and avoidance of premature entry into S phase. These include the of securin and cyclin B, which depends on the completion of mitotic cyclins, which continue to be translated after exit from chromosome bi-orientation, the APC/C-dependent degradation of mitosis, but are unstable in G1 phase (Almeida et al., 2005), and cyclin A occurs as soon as cells enter mitosis, despite the activity the F-box protein SKP2, which controls the abundance of the CDK of the mitotic checkpoint (den Elzen and Pines, 2001; Geley et al., inhibitor p27Kip1 (Wei et al., 2004). Recently, it has been shown that 2001). These differences are poorly understood, but recent evidence the tumour suppressor RB1 (pRB) cooperates with FZR1 in targeting points to significant differences in the molecular recognition of SKP2 for polyubiquitylation (Binne et al., 2007). Thus, in addition different substrates (Kimata et al., 2008; Matyskiela and Morgan, to its role in destabilising late mitotic substrates, FZR1 seems to 2009; Wolthuis et al., 2008). have an important role in controlling G1 phase. By maintaining the As cyclins are degraded, CDK activity declines and phosphatases APC/C activity during exit from mitosis and in early G1 phase, FZR1 convert the APC/C back to its hypophosphorylated interphase state, could also contribute to the establishment of pre-replicative which is activated by Fizzy-related (FZR1, also known as CDH1), complexes (pre-RCs) on origins of DNA replication by providing a a CDC20-related protein (Kramer et al., 1998). Similarly to CDC20, time window with low CDK activity (reviewed by Diffley, 2004). FZR1 contains seven WD40 repeats, which form a seven-bladed At the end of G1 phase, the APC/C is inactivated by FBXO5 propeller that is involved in substrate and APC/C binding (Kraft et (Emi1), which binds to and inactivates FZR1, allowing cyclin A to FZR1 function in mitosis and G1 phase 4209

accumulate and activate CDK2. Active CDK2, in turn, contributes conditional gene ablation in the mouse. In both systems, we found to the inactivation of the APC/C by inhibitory phosphorylation of that cells had no obvious problems in returning to interphase after FZR1 (Kramer et al., 2000). In addition, APC/C activity in G1 phase the end of mitosis. Cells lacking FZR1, however, had an unusually is self-limited by autoubiquitylation and degradation of UBCH10, short G1 phase and proliferated poorly. one of the E2 enzymes of APC/C (Rape and Kirschner, 2004). Knockdown of UBCH10 has recently been shown to shorten G1 Results phase by increasing cyclin A levels, suggesting that UBCH10 is Human FZR1 is not required for exit from mitosis but controls the essential E2 for FZR1-APC/C in G1 phase (Rape and Kirschner, the onset of DNA replication 2004; Walker et al., 2008). To study whether FZR1 is essential for exit from mitosis, we used FZR1 and its orthologues have been studied in several transient RNAi to knockdown FZR1 in human U2OS organisms and were found to be important for establishing or osteosarcoma cells, which reduced FZR1 levels to more than 95% maintaining G1 phase, but are dispensable for mitosis (Blanco et as determined by immunoblotting and densitometry scanning. Fig. al., 2000; Jacobs et al., 2002; Sudo et al., 2001; Visintin et al., 1A shows that knockdown of FZR1 stabilised several known FZR1 1997). By contrast, analysis of mouse embryonic fibroblasts substrates, such as hKid (KIF22), Aurora kinase A (AURKA) and (MEFs) derived from FZR1-deficient knockout mice has provided B (AURKB). However, CDC20 and PLK1 were only slightly evidence that FZR1 is involved in regulating exit from mitosis stabilised in FZR1 RNAi cells released from a nocodazole (noc) (Garcia-Higuera et al., 2008; Li et al., 2008), whereas FZR1 RNAi- block. Their overall levels in RNAi cells were higher and both based studies in human cells have been controversial (Floyd et proteins re-accumulated faster in late-G1 phase than in controls. al., 2008; Engelbert et al., 2008). In FZR1 RNAi cells, the early-G1 phase decline of CDC25A and To clarify the role of FZR1 during exit from mitosis, we used UBCH10 was prevented, but as cells progressed towards S phase, two experimental systems using RNAi in human cells and the protein levels of both proteins dropped. SKP2 declined in G1-S Journal of Cell Science

Fig. 1. FZR1 RNAi in human cells does not affect exit from mitosis. (A)U2OS cells transfected with control and FZR1 siRNAs were synchronised in mitosis (M) and released for analysis of APC/C substrates by immunoblotting. FZR1 substrates are indicated by a, mitotic APC/C substrates by b, and G1-phase substrates by c. (B)Signals were scanned and plotted normalised to GAPDH values. (C)Length of mitosis determined by videomicroscopy of U2OS FZR1 RNAi or control cells. (D)U2OS cyclin B1-GFP cells were induced for 24 hours with 1g/ml dox, transfected with FZR1 siRNA for 24 hours and monitored by fluorescence microscopy. Arrows highlight a dividing cell, arrowheads G1-phase cells. Scale bar: 20m. Time is hours:minutes. (E)Stable inducible FZR1 RNAi cells were induced for 3 days using 1g/ml dox, arrested in mitosis, harvested, released and analysed for cyclin B1, FZR1 and GAPDH. (F)FZR1 RNAi cells treated with or without doxycyline (dox) were released from a mitotic block, stained for AURKB, - tubulin and DNA and evaluated for bi-nucleation. 4210 Journal of Cell Science 122 (22)

phase independently of FZR1 levels (Fig. 1Ac,1B) but the overall amount of SKP2 was about ten times higher in FZR1 RNAi cells. In contrast to the above FZR1 substrates, the degradation of cyclin A and cyclin B1, geminin and securin was not affected in mitosis (Fig. 1B), but these proteins re-accumulated ~2 hours sooner in G1 phase in FZR1-knockdown cells (Fig. 1B). In summary, knockdown of FZR1 only affected the degradation of late mitotic and G1-phase APC/C substrates. But although late mitotic substrates, such as AURKA and PLK1 were stabilised, the length of mitosis was not significantly prolonged, as judged as the time from cell rounding to initiation of cytokinesis by live-cell phase-contrast time-lapse microscopy (Fig. 1C). To monitor cyclin degradation with higher resolution, U2OS cells with tetracycline-inducible expression of cyclin-B1-GFP (Wolf et al., 2006) were grown in the presence of 1 g/ml doxycyline (dox), transfected with 50 nM FZR1 siRNA for 24 hours and analysed by fluorescence time-lapse imaging. As Fig. 1D shows, cyclin B1-GFP was efficiently degraded during mitosis in the absence of FZR1 but re-accumulated faster in G1 phase [2.4±0.46 hours after cytokinesis in RNAi cells (n8) compared with 5.5±1.3 hours in control cells (n15)]. Similar effects of transient FZR1 knockdown on APC/C substrate stabilisation and exit from mitosis were obtained in HeLa cervical carcinoma cells (data not shown). To determine the long-term effects of FZR1 knockdown, we established a line of U2OS cells with dox-inducible expression of a short hairpin RNA (shRNA) directed against FZR1. Induction of this shRNA caused a more than 95% reduction of FZR1 protein levels (supplementary material Fig. S1) and inhibited the degradation of AURKA, AURKB and hKid (not shown). As in the above experiments performed with transient transfection of siRNAs, the degradation of cyclin B1 during mitosis was not affected in Fig. 2. FZR1 RNAi impairs proliferation and shortens G1 phase. (A)Cell induced cells released from a mitotic arrest (Fig. 1E). Consistently, numbers of FZR1 RNAi clones treated with or without dox were determined the length of mitosis (mean duration 32±5.5 minutes, n107, in over 8 days and cell extracts analysed for FZR1 expression. (B)Competitive non-induced controls and 37±17.8 minutes, n161, in FZR1- growth analysis was performed by mixing equal numbers of inducible GFP- positive FZR1 RNAi (U2OS-FZR1-shRNA) cells with parental U2OS cells in

Journal of Cell Science knockdown cells) was not altered. In addition, using the presence or absence of inducer (doxycyline, dox) and GFP-positive cells immunofluorescence microscopy, we could not detect a higher were counted over five passages. shRNA ± dox indicates percentage of GFP- frequency of cytokinesis failure in FZR1-knockdown cells, in which positive cells in U2OS-FZR1-shRNA cells cultivated alone. (C)Long-term AURKB was stabilised (Fig. 1F). In summary, FZR1 RNAi, cultures of induced and non-induced FZR1 RNAi cells were analysed for induced by transient siRNA transfection or inducible expression of protein expression. (D)Conditional FZR1 U2OS cells were induced for up to shRNAs in stable cell lines, did not hamper progression through 12 days with doxycycline and the fate of daughter cells monitored for 36 hours by videomicroscopy., mean generation time in hours. (E)U2OS cells mitosis. transfected with control or FZR1 siRNA for 24 hours were nocodazole- When conditional FZR1 RNAi U2OS cells were treated for synchronised in mitosis and assayed for BrdU incorporation in released cells. prolonged periods with 1 g/ml dox they were found to grow more (F)Conditional FZR1 cells were induced with 1g/ml dox for 3 days, arrested slowly than controls (Fig. 2A). To confirm that knockdown of FZR1 in mitosis with nocodazole, and released into G1 phase by mitotic shake-off and replating. Cells were harvested at the indicated time points and analysed impairs proliferation, we mixed GFP-expressing FZR1 shRNA cells for DNA content by PI staining and flow cytometry. (G)U2OS cells were with their parental cell line (U2OS) in the presence or absence of transfected as in E and filmed for 16 hours using phase-contrast microscopy dox and measured the proportion of GFP-positive RNAi cells by before addition of 10M BrdU for 1 hour, followed by BrdU staining. flow cytometry. As can be seen in Fig. 2B, RNAi induction with Individual cells were tracked from their birth (completion of cytokinesis) until dox caused a proliferation disadvantage and percentage loss of GFP- the end of the experiment and evaluated for BrdU incorporation. positive FZR1 RNAi cells (MIX+dox). When stable FZR1- knockdown cells were cultivated for up to 11 days and analysed by immunoblotting, we noted that FZR1 RNAi led to an increase generation time (µ) + 2 s.d.], suggesting that they might have of cyclin B1 levels (Fig. 2C) and strong induction of the CDK withdrawn from the cell cycle. By contrast, in non-induced control inhibitor p21CIP1, starting at day 3 of shRNA induction. cells only 1.9% of cells failed to divide within the observation To determine the cause underlying the reduced growth rate, we period. monitored conditional FZR1 RNAi cells in the presence or absence of dox by time-lapse phase-contrast microscopy. As can be seen in FZR1 is required to establish and maintain G1 phase Fig. 2D, the generation time increased from 19.6±2.27 hours in Because mitotic cyclins, in particular cyclin A, re-accumulated faster control cells to 23.2±4.05 hours in FZR1-knockdown cells treated in the G1 phase of FZR1 RNAi cells, we hypothesised that FZR1 for 6 days with dox. In addition, in response to FZR1 knockdown, RNAi might induce premature entry into S phase, which has been up to 13% of cells failed to divide within 31 hours [ mean suggested to induce replication stress and DNA-damage responses FZR1 function in mitosis and G1 phase 4211

(Bartek et al., 2007), leading to p21CIP1 induction or cell death. To have a more synchronous population of FZR1 RNAi cells, we transiently transfected U2OS cells with 60 nM control or FZR1 siRNAs for 24 hours before addition of 250 nM noc for 14 hours. Cells were then synchronously released from the mitotic block and stained for BrdU incorporation every hour. This revealed that half of the FZR1 RNAi cells were already BrdU positive at 4 hours after release, whereas it took control cells 7-8 hours to reach the same number of positive nuclei (Fig. 2E). Similarly, when conditional FZR1 U2OS cells were induced with dox and analysed for cell cycle progression by DNA staining and flow cytometry after release from a mitotic block, a faster accumulation of S-phase cells could be observed (Fig. 2F). Next, we asked whether FZR1 is required for establishing G1 phase in serum-starved cells. Induced and non-induced U2OS FZR1 RNAi cells were cultivated in the absence of growth factors and pulse labelled with BrdU. FZR1-knockdown cells were found to contain a higher number of BrdU-positive cells (51.6%) than controls (27.7%) and serum-restimulated cells entered S phase faster (supplementary material Fig. S2). Thus, FZR1 is required for establishing G1 phase and determines its length. To analyse whether the faster onset of DNA replication was due to premature CDK activity, cyclin A was immunoprecipitated from Fig. 3. FZR1 RNAi induces a DNA-damage-response pathway. (A)Inducible control and FZR1 RNAi cells released from mitotic block. FZR1 RNAi cells were grown for 5 days with or without 1g/ml dox and exposed to 8 Gy gamma irradiation or left untreated. Total cell lysates obtained Associated kinase activity started to increase at 2 hours after release at the indicated time points were analysed by immunoblotting. The asterisk in in FZR1 RNAi but not in control cells (supplementary material Fig. the CHK2 blot indicates a non-specific band. (B)Conditional FZR1 RNAi, and S3), suggesting that precocious activation of cyclin A or CDK2 FZR1-p53 double RNAi U2OS cells were treated with dox and analysed for could trigger the onset of S phase. To test this hypothesis directly, protein expression of total cell lysates obtained at the indicated time points. (C)Cell numbers of cultures treated for the indicated time points with dox are we measured the onset of DNA replication in cell lines expressing shown relative to uninduced control cells. a mutant of cyclin A (lacking amino acid residues 47-83, encompassing the D-box) (Geley et al., 2001). When cells induced for expression of cyclin A 47-83 were released from a noc block, ten times more BrdU-positive nuclei (30.2%; n192) could be RNAi cells spent more time in S phase. An increase in S-phase detected at 7 hours than in non-induced control cells (3.2%; n94). duration was also seen when asynchronous cells were pulse-

Journal of Cell Science Thus, premature accumulation of cyclin A is sufficient to trigger labelled with BrdU, which revealed that a higher fraction (56%) of DNA replication, which establishes cyclin A as an essential target FZR1-knockdown cells were in S phase compared with 45% in of FZR1-APC/C in G1 phase, consistent with previous findings controls (n>1000 in each experiment). using cells in which levels of UBCH10 were reduced by RNAi (Walker et al., 2008). FZR1 RNAi activates the DNA-damage response and induces a cell-type-specific phenotype Loss of FZR1 function causes prolongation of S phase The prolongation of S phase and the induction of p21CIP1 in FZR1 The above experiments revealed that FZR1 knockdown not only RNAi cells prompted us to investigate whether knockdown of FZR1 caused a shortening of G1 phase but also a gradual increase in activated the DNA-damage response pathway. As can be seen in generation time. Because DNA content analysis by flow cytometry Fig. 3, U2OS cells -irradiated with 8Gy rapidly produced did not reveal an increase in G2-M cells in FZR1 RNAi cells (Fig. phosphorylated histone H2AX and CHK2, substrates of the 2F, supplementary material Fig. S1B, Fig. S4A), we determined ATM/ATR kinases. In addition, p53 exhibited retarded mobility, the length of S phase in FZR1 RNAi cells. Mock-transfected and suggesting activation by phosphorylation. Consistently with p53 FZR1 siRNA-transfected U2OS cells were filmed for 16 hours by activation, the CDK inhibitor p21CIP1 was strongly induced in - time-lapse microscopy and pulse-labelled with BrdU for 1 hour irradiated cells, whereas the levels of phospho-histone H3 declined before fixation and staining. Analysis of movies allowed us to because of inhibition of mitosis. When dox-induced FZR1 RNAi correlate the age of individual cells (with respect to their previous cells were analysed, it became evident that several of the DNA- division) with the onset of DNA replication. This analysis revealed damage response markers, such as phospho-H2AX, p53 and p21CIP1, that in some FZR1 RNAi cells, DNA synthesis was initiated within were already expressed, even without -irradiation. In contrast to the first hour after completion of cytokinesis, and more than 50% irradiated cells, however, FZR1 RNAi U2OS cells did not suppress of the cells were BrdU positive after ~3 hours. In control cells phospho-histone H3 and continued to proliferate (see also (expressing FZR1), the first BrdU-positive cells appeared more than supplementary material Fig. S1B). 4 hours after cytokinesis and 50% of the cells replicated their DNA The above data suggested that loss of FZR1 function activated only after 8 hours. In FZR1 RNAi cells the number of BrdU-positive p53 and subsequent induction of p21CIP1 and cell death, at least in nuclei older than 4 hours was high and, as in control cells, only some FZR1 RNAi U2OS cells. To investigate the role of p53, we declined in cells that had undergone cytokinesis more than 14 hours first analysed the effect of FZR1 RNAi in different human cell lines earlier. Thus, although DNA replication was initiated earlier, FZR1 with wild-type or altered p53 function, including HeLa (p53 4212 Journal of Cell Science 122 (22)

inactivated) and Hct116 (colon carcinoma, p53 wild-type) tumour To further investigate the role of p53 in the antiproliferative effect cells, p53 wild-type non-transformed MRC5 fibroblast and RPE1 of FZR1 RNAi, we stably expressed a p53-targeting shRNA in retinal pigment epithelial cell lines. In contrast to U2OS cells, which conditional FZR1 U2OS cells. Induction of FZR1 RNAi in these mainly delayed progression through S phase upon reduction of cells failed to induce p21CIP1 (Fig. 3B), demonstrating that p53 was FZR1 levels, MRC5 and RPE1 cells responded with a strong G1 effectively silenced. Although stabilisation of cyclins, or phospho- arrest after 6 days of dox treatment. Long-term cultured HeLa and H2AX induction were not affected (not shown), p53 knockdown Hct116 FZR1 RNAi cells also accumulated in G1 phase, but in could not rescue the proliferation disadvantage of FZR1 RNAi cells addition underwent cell death and displayed a sub-G1 DNA content (Fig. 3C). Thus, the p53 response is a consequence of the premature that was characteristic of apoptosis (supplementary material Fig. entry into, and impaired progression through, S phase, which is S4). In all conditional FZR1 RNAi cell lines, p21CIP1 and p27KIP1 caused by knockdown of FZR1. were strongly induced, as was phospho-H2AX. Although FZR1 RNAi impaired proliferation in all cellular model systems, the Mouse FZR1 is not required for exit from mitosis but controls different cellular responses might be due to cellular differences in G1 phase p53 function or their ability to activate cell cycle checkpoints in To determine the role of FZR1 in mammalian cells without the response to a premature entry into S phase. potential ambiguities associated with RNAi approaches, we Journal of Cell Science

Fig. 4. Generation and analysis of Fzr1-deficient mouse embryonic fibroblasts. (A)Fzr1 targeting strategy. The 5Ј homology arm of the Fzr1-targeting vector contains a neomycin resistance (Neo) and a kanamycin resistance (K) gene flanked by Frt sites, along with a loxP site in the intron 1 sequences of the Fzr1 gene. The second loxP site was integrated into intron 8. The plasmid backbone carries a thymidine kinase (TK) negative selection marker as well as an ampicillin resistance gene (A). Restriction enzyme sites for EcoRI and HinDIII are indicated, as are the locations of the probes for Southern hybridisations. Arrows show the positions of the primers for PCR genotyping to detect wild-type (Fzr1wt) and floxed (Fzr1fl) using PCR1, and the exon 2-8 deleted allele (Fzr1–) by PCR2. (B)Embryonic stem cell clones were screened by Southern blotting using DNA digested with HindIII and EcoRI, with probes SP2 and SP1, respectively. (C)PCR genotyping of mice for the presence of the loxP site in intron 2 (PCR1) and for the deletion of exon 2 to exon 8 (PCR2). (D)Cell numbers of control (Fzr1fl/fl) and Fzr1-deficient (Fzr1–/–) MEFs were determined for 7 days (mean ± s.d.). (E)Control (Fzr1fl/fl) and Fzr1-deficient (Fzr1–/–) MEFs were monitored by videomicroscopy and duration of mitosis was scored from the onset of ‘rounding up’ until completion of cytokinesis. Selected 5-minute interval frames from cells undergoing mitosis of control (Fzr1fl/fl) and Fzr1–/– MEFs are shown. Scale bar: 20m. FZR1 function in mitosis and G1 phase 4213

generated a mouse strain harbouring a conditional Fzr1 allele (Fig. In order to determine whether Fzr1 is essential at the cellular 4A) based on the loxP/Cre recombination system. FZR1 is level, we obtained mouse embryonic fibroblasts (MEFs) from encoded by 14 exons located in the C1 region of chromosome embryonic day 12 embryos of homozygous Fzr1flNeo/flNeo mice and 10. By using a ‘recombineering’ strategy (Zhang et al., 2002), immortalised them at passage 6 by transduction with a SV40 large we generated a gene-targeting vector (Fig. 4A) to introduce loxP- T-expressing retrovirus. After expansion, cells were infected with site-specific recombination sequences into intron 2 and intron 8, a Cre-recombinase expressing adenovirus to delete Fzr1 exon 2 to respectively. Deletion of exon 2 to exon 8 by Cre recombinase exon 8, which was verified by PCR and Southern blotting. The removes the translation start codon as well as the coding established Frz1-proficient and Frz1-deficient cell lines are from sequences for the conserved C-box region (Yu, 2007) and the now on referred to as Fzr1fl/fl (control) and Fzr1–/– cells, first two WD40 domains of FZR1. Correctly targeted R1 respectively. In comparison to control cells (infected with a YFP- embryonic stem (ES) cell clones were identified by Southern expressing adenovirus), FZR1 was not detectable by blotting (Fig. 4B), and clones 3A3 and 4F1 used for blastocyst immunoblotting in Fzr1–/– cells (Fig. 5A). Fzr1fl/fl and Fzr1–/– microinjection to obtain chimeric mice with germline SV40-large-T-immortalised MEFs proliferated with similar kinetics transmission. After establishing Fzr1flNeo/flNeo mice (Fig. 4C), the (Fig. 4D), demonstrating that Fzr1 is not essential for proliferation, neomycin resistance cassette was genetically deleted to obtain at least in SV40-large-T-immortalised cells. In addition, by Fzr1fl/fl mice, which were found to be healthy and fertile. monitoring the length of mitosis in Fzr1fl/fl and Fzr1–/– cells, we Deletion of Fzr1 exon 2 to exon 8, by crossing to PGK-Cre found that exit from mitosis was not significantly delayed in cells transgenic mice, however, confirmed the previously reported lacking FZR1 (Fig. 4E). embryonic lethality (Garcia-Higuera et al., 2008; Li et al., 2008) Consistent with the normal length of mitosis, the degradation and no homozygous Fzr1-deficient mice could be detected of mitotic cyclin B1 and PLK1 was not delayed by the absence among 73 (27 Fzr1+/+, 46 Fzr1+/–, 0 Fzr1–/–) offspring from of FZR1 (Fig. 5A,B). Similar results were obtained in cyclin B1 pairings of heterozygous Fzr1+/– mice. immunostaining experiments, which revealed that cyclin B1 Journal of Cell Science

Fig. 5. Loss of FZR1 does not affect exit from mitosis. (A)Control (Fzr1fl/fl) and Fzr1–/– MEFs were synchronised in mitosis, released and harvested at the indicated time points (minutes) for evaluation of APC/C substrates by immunoblotting. (B)After densitometry, signals were normalised to PRC1 signals, which served as a loading control, and plotted. (C)Control and Fzr1–/– MEFs were stained for cyclin B1, pericentrin and DNA. Scale bar: 10m. (D)Quantification of cyclin B1 intensity levels on centrosomes at various stages of mitosis in control (white bars) and Fzr1-knockout MEFs (black bars). (E)Control and Fzr1–/– MEFs were stained for AURKB, -tubulin and DNA. (F)Control and Fzr1–/– MEFs were released from a nocodazole block and analysed for AURKB signals and binucleated cells. 4214 Journal of Cell Science 122 (22)

exit from mitosis in murine cells, and this decline was not affected by loss of FZR1 function. Mitotic cyclins were degraded with similar kinetics in wild-type and Fzr1-deficient MEFs (Fig. 5A, Fig. 6B). When APC/C was immunoprecipitated from nocodazole-treated arrested and released cells using a CDC27-specific antibody, the amount of co-immunoprecipitated CDC20 decreased in control cells but was maintained for longer in Fzr1-deficient cells (Fig. 6C, arrows). Thus, in the absence of FZR1, more CDC20 appeared to be bound to the APC/C, possibly explaining why the degradation of late mitotic substrates, such as PLK1, occurred normally in cells lacking Fzr1. As we observed in human cells depleted of FZR1 by RNAi, mitotic cyclin A and cyclin B reaccumulated earlier during G1 phase in cells of Fzr1-deficient mice. In addition, the oscillation of Skp2 was prevented and Fzr1-deficient cells exhibited a premature decline of p27Kip1 (Fig. 6B). When control and Fzr1-deficient MEFs were stained for cyclin A, 56% and 74% of cells were positive, respectively, suggesting an increase of Fzr1-deficient cells in S phase. BrdU incorporation in synchronised cells released from a nocodazole block confirmed this, and demonstrated a considerable shortening of G1 phase in Fzr1-deficient MEFs, as well as an increase in the duration of S phase (Fig. 6D). When MEFs were starved of growth factors for 3 days and analysed for S-phase cells, Fzr1-deficient cells contained more BrdU-positive nuclei (45.1%; n226) than controls (21.8%; n220), indicating a reduced capacity to arrest in G1 phase. In summary, our data show that FZR1 is not required for mitosis, but for establishing G1 phase and efficient progression through S phase.

Discussion APC/C-dependent proteolysis is essential for progression through Fig. 6. Loss of FZR1 causes stabilisation of APC/C substrates in G1 phase and mitosis. In all organisms studied, CDC20 and core APC/C subunit fl/fl –/– promotes entry into S phase. (A)Control (Fzr1 ) and Fzr1 MEFs were are essential genes and cells lacking APC/C activity arrest synchronised by noc (M) and released for analysis of APC/C substrates by immunoblotting. (B)Immunoblots from A were scanned and evaluated by at metaphase (Nasmyth, 2002; Wirth et al., 2004). CDC20 is,

Journal of Cell Science densitometry. (C)Cell lysates obtained from noc-released MEFs at the however, not the only activator of the APC/C and several CDC20- indicated time points were used for CDC27 immunoprecipitation and related proteins have been described. One of these conserved immunoblotting for CDC20 and FZR1. Arrows indicate the 2-hour release proteins is FZR1, which is kept inactive by CDK-dependent time point of CDC27 immunoprecipitates. (D)Control and Fzr1–/– MEFs were filmed by phase-contrast microscopy for 16 hours, incubated for 1 hour with phosphorylation but becomes active after the initiation of cyclin 10M BrdU and stained for BrdU incorporation. Cellular age was plotted as a degradation during anaphase. Although FZR1 has been implicated function of the percentage of BrdU-positive cells. in the regulation of mitosis in budding yeast (Schwab et al., 1997) and Drosophila (Raff et al., 2002), it is not essential for exit from mitosis in these organisms. levels were similar at various stages of mitosis, regardless of the As cells exit mitosis, proteins such as AURKA, AURKB and Fzr1 genotype (Fig. 5C,D). Because cytokinesis is highly PLK1 (Pines, 2006) are degraded in a FZR1-APC/C-dependent sensitive to even low amounts of any remaining cyclin B1 (Wolf manner, but the significance of this degradation is only poorly et al., 2006), we stained cells for AURKB, microtubules and DNA understood. When cells are blocked at metaphase by inhibition of (Fig. 5E) and counted bi-nucleated cells after release from a the proteasome, exit from mitosis can be triggered by the sole nocodazole block. As can be seen in Fig. 5F, no increase in inhibition of CDK activity (Potapova et al., 2006). Thus, it was binucleated cells could be detected in Fzr1-deficient cells, which unclear whether degradation of any substrates, except securin and lacked APC/C activity, as shown by the stabilisation of AURKB. the mitotic cyclins, is required for mitosis and this motivated our Thus, our data demonstrate that, as in human RNAi cells, FZR1 study. We have approached this problem by targeting FZR1 in is not required for exit from mitosis in immortalised mouse mammalian cells by using RNAi in human cells as well as by genetic embryonic fibroblasts. deletion in the mouse, and show that FZR1 is not required for Next, we analysed the stability of APC/C substrates by completion of mitosis. We have carefully analysed the effect of immunoblotting cell extracts prepared from synchronised cell FZR1 depletion on the degradation of cyclin B1, which needs to cultures of control or Fzr1-deficient MEFs. As can be seen in Fig. be degraded completely for cells to be able to leave mitosis (Wolf 6A, FZR1 substrates such as KID (KIF22), AURKB, CDC20 and et al., 2006). In both human RNAi cells and Fzr1-deficient MEFs, B99 were stabilised. Similarly to human RNAi cells, PLK1 was we could not detect an effect of FZR1 depletion on cyclin only slightly stabilised in the absence of FZR1 and similarly to degradation in mitosis. Our results agree with that of others (Floyd CDC20, reaccumulated faster in G1 phase. In contrast to human et al., 2008), who have also shown that FZR1 is not essential for U2OS cells, however, PLK1 levels dropped more sharply during mitosis. FZR1 function in mitosis and G1 phase 4215

Other reports, however, described that loss of FZR1 function the generation time and permanent withdrawal from the cell cycle, delayed exit from mitosis and induced mitotic defects (Engelbert as well as the induction of cell death, possibly explaining the et al., 2008; Garcia-Higuera et al., 2008). We believe that these different cellular phenotypes observed in FZR1 RNAi cells. The effects are consequences of the genetic damage caused by FZR1 failure of p53 depletion to restore the proliferative capacity in FZR1 depletion. In contrast to the lack of an effect on mitosis, loss of knockdown U2OS cells, however, demonstrates that loss of FZR1 FZR1 severely affected G1 phase. The shortening of G1 phase might function has a more profound effect on cell cycle progression, most be caused by the premature accumulation of cyclin A, stabilisation likely by inducing genetic alterations that are incompatible with of SKP2 and CDC25A, all of which are known to be involved in efficient proliferation. the regulation of the G1-S transition (Pagano, 2004). Enforced entry In summary, by analysing the degradation of a large set of known into S phase might adversely affect the mechanics or fidelity of APC/C substrates in cells lacking FZR1 function, we found that DNA replication and cause replication stress, which has been shown proteolysis was only affected for proteins that are normally degraded to induce DNA-damage responses (reviewed by Branzei and Foiani, in late mitosis or in G1 phase. Failure to degrade these late mitotic 2009). Thus, the reported increase in binucleated and abnormal substrates, however, did not interfere with completion of mitosis. mitoses in FZR1-deficient cells (Engelbert et al., 2008; Garcia- In contrast to CDC20, there appears to be no essential FZR1 Higuera et al., 2008) might be caused by lagging or broken substrate that has to be degraded during each and every cellular that have interfered with the completion of division. FZR1, however, controls the abundance of important G1- cytokinesis. In addition, a lack of FZR1 might also weaken the phase regulators, including SKP2, CDC25A and cyclin A, and is DNA-damage and DNA-replication checkpoints by stabilisation of needed for establishing G1 phase and the timing of DNA replication. PLK1 (Bassermann et al., 2008), which would further promote Thus, the ability of FZR1 to flush remaining mitotic proteins from genetic instability by facilitating entry into mitosis (Garcia-Higuera the cell is less important than its function in controlling their et al., 2008). reappearance in the daughter cells, and it will be important to Fzr1-deficient cells prematurely initiate DNA replication and determine this role in vivo using tissue-specific deletion of Fzr1. spend a longer time in S phase. In addition, they fail to arrest in G1 phase after serum starvation, suggesting that G1 phase APC/C Materials and Methods activity sets a threshold for growth factor stimulation, as has also Reagents Chemical reagents were obtained from Sigma, enzymes from Promega (Mannheim, been reported in primary cells obtained from Apc2-deficient mice Germany) and DNA oligonucleotides from MWG Biotech (Ebersberg, Germany), (Wirth et al., 2004). The prolongation of S phase might have trivial unless stated otherwise. Antibodies used in this study were AURKA (rb, 12875), causes, such as the lack of sufficient factors required for replication, AURKB (rb, 2354), CDC25A (m, 2357), PLK1 (m, 14210), Securin (m, 3305), such as nucleotides, owing to the shortening of a cellular growth p21Cip1 (rb, Ab7960) from Abcam (Cambridge, UK); B99 (rb, from Claudio Schneider, LNCIB, Trieste, Italy), CDC20 (rb, Sah107, from Jan-Michael Peters, period during G1 phase. Shortening of one cell cycle phase is known Institute for Molecular Pathology, Vienna, Austria), CDC27 (m, AF3.1, from Julian to cause compensatory lengthening of other phases (Reis and Edgar, Gannon, CRUK, Clare Hall Laboratories, South Mimms, UK and BD Transduction 2004). The increase in the duration of S phase is, however, unlikely Labs, Schwechat, Austria), Cyclin A [m, E23 (Julian Gannon), SG20 (raised against the N-terminal 170 amino acids of human cyclin A)], Cyclin B1 (m, V152, Julian to be just a compensatory mechanism for the reduction of G1 phase, Gannon), GAPDH (m, 6C5, HighTest), Geminin (r, 8B1, from Aloys Schepers, because all the conditional FZR1 RNAi cell lines displayed signs Helmholtz Zentrum, Munich, Germany), KID [m, 8C12 and rb CW3, raised against

Journal of Cell Science of severe cellular stress and either slowed down proliferation or the C-terminal 250 amino acid residues of hKID (Wandke and Geley, 2006)], p27KIP1 even induced cell death. (rb, Santa Cruz Biotechnology, Heidelberg, Germany), SKP2 (m, Zymed), UbcH10 (rb, Boston Biochem, Cambridge, MA), phospho-histone H3 (rb, Upstate, Millipore, Shortening of G1 phase and forced entry into S phase might limit Vienna), p-Chk2 (rb, PA1-14093, Affinity Bio Reagents, Thermo Scientific), p53 (m, the number of functional pre-RCs or might lead to replication fork pAb1801, Oncogene Science), p-H2AX (rb, 9718, Cell Signaling, NEB). FZR1 (m, collapse, as suggested previously (Bartkova et al., 2006; Di Micco AR38, from Julian Gannon, raised against recombinant FZR1), FZR1 (Rb, raised against an N-terminal peptide, affinity purified). To obtain monoclonal FZR1 et al., 2006). Because neither mitotic cyclins nor geminin oscillations antibodies, full-length human FZR1 coding sequence was subcloned into bacterial were affected by FZR1 RNAi or Fzr1 deletion, the replication expression vector pET28a (Novagen, Merck Chemical, Nottingham, UK) and licensing cycle did not seem to have been affected. Levels of CDC6, the recombinant C-terminally hexa-histidine-tagged FZR1 expressed in BL21[DE3]pLysS, purified from inclusion bodies under denaturing conditions and which is reported to be an APC/C substrate (Mailand and Diffley, used to immunise Balb/c mice as described (Wandke and Geley, 2006). Spleen cells 2005), did not oscillate during the cell cycle in the cell systems were used for fusion with Sp2 myeloma cells and clones screened for antibodies by used in this study. Thus, the re-setting of the cell cycle clock was Elisa. Clone AR38 was expanded and found suitable for immunoblotting but not for not obviously abrogated by loss of FZR1 and a short (but perhaps immunostaining or immunoprecipitation. For production of polyclonal rabbit anti- FZR1 antibodies, anti-peptide antibodies (using the N-terminal 18 residues as not quite adequate) time window for establishing pre-RCs was antigen) were produced by Gramsch Laboratories (Schwabhausen, Germany) and generated. It is not currently known whether it is the number, affinity purified using the immunogen coupled to thiopropyl-Sepharose-6 beads (GE- activation or set-up of pre-RCs, or whether it is replication fork Healthcare, Vienna, Austria). movement that is perturbed in cells lacking FZR1 function. The Plasmids cellular model systems that we have established, however, should The inducible lentiviral FZR1 RNAi vectors were generated by LR-clonase be useful in determining whether and how FZR1 affects origin (Invitrogen, Lofer, Austria) mediated transfer of a short-hairpin RNA (shRNA) expression cassette targeting human FZR1 (Brummelkamp et al., 2002) from pENTR- licensing (Sivaprasad et al., 2007) and DNA replication. THT into the lentiviral vectors pHR-DEST-SFFV-GFP and pHR-DEST-SFFV-PURO In Fzr1-deficient MEFs transduced with SV40-LT, S phase is (Ploner et al., 2008). For constitutive expression of shRNAs, the H1 promoter from also prolonged, but cellular proliferation is not affected. By contrast, pRETRO-Super (kindly provided by Reuven Agami, The Netherlands Cancer primary MEFs lacking FZR1 (Garcia-Higuera et al., 2008; Li et Institute, Amsterdam, The Netherlands) was subcloned into pENTR-1A (Invitrogen) using EcoRI and SalI. A 64bp p53 targeting (underlined sequence) shRNA al., 2008) proliferate poorly and undergo premature senescence. This encoding oligonucleotide (5Ј-GATCCCCAGTAGATTACCACTGGAGTCttcaagaga - suggests that LT interferes with a cellular response, such as GACTCCAGTGGTAATCTACTTTTTTGGAAA-3Ј) was subcloned using BglII and activation of p53, which impairs proliferation in the absence of HindIII and sequenced. Lentiviral constructs for delivery of the shRNA expression cassette were done using GATEWAY technology. Adenoviral constructs were created FZR1 function. Depletion of FZR1 by RNAi in human cells also by shuttling the iCRE- or Venus-encoding DNA fragments from corresponding ENTR activates a p53 response, which could explain the lengthening of vectors into pAD/CMV-DEST (Invitrogen). 4216 Journal of Cell Science 122 (22)

Cell lines, virus production and RNAi at 4°C with SG20 antibodies covalently coupled to Affiprep protein-A beads. After U2OS (ATTC:HTB-96), HEK293T, HEK293A (Invitrogen), RPE1 (kindly provided three washes in lysis buffer, beads were split for immunoblotting and kinase by Erich A. Nigg, Max-Planck-Institute of Biochemistry, Martinsried, Germany), reactions. For kinase reactions, beads were washed once in kinase buffer (3 mM MRC-5 (ECACC, Salisbury, UK) and HCT116 (ATTC:CCL-247) cells were grown EGTA, 5 mM NaF, 50 mM -glycerophosphate, 1 mM DTT and 15 mM magnesium in DMEM supplemented with 10% FCS, 100 g/ml streptomycin and 100 U/ml acetate) and incubated with 250 g/ml histone H1 and 5 Ci [-32P]ATP for 20 minutes penicillin in saturated humidity at 37°C, 5% CO2. For synchronisation, cells were at 30°C. Kinase reaction were stopped on dry ice and analysed by denaturing gel treated for 12-14 hours with 250 nM noc and harvested by mitotic shake-off, washed electrophoresis and autoradiography. Quantification was carried out by densitometric three times in medium and replated. For BrdU labelling, cells were incubated for 1 scanning of exposed films and analysis by ImageJ software (Abramoff et al., 2004). hour in 10 M BrdU. Cell lines (U2OS, HeLa, Hct116, MRC-5, RPE1) expressing TetR (U2OS-TR) were generated by lentiviral transduction using pLENTI6-TR We are very grateful to Julian Gannon for antibodies; Steve Elledge (Invitrogen). MEFs were obtained from fetuses at E12 according to standard protocols. MEFs were grown in 50:50 DMEM Ham’s F-12 medium supplemented (Harvard Medical School, Boston, MA) and Neal G. Copeland (NCI as above and immortalised by transduction with a SV40-TAg-expressing retrovirus Frederick, MD) for gene-targeting reagents; Reuven Agami for obtained from Y2-865 cells. Production of retrovirus, lentivirus and adenovirus was pRETRO-SUPER; Ian Rosewell (CRUK, Clare Hall) for ESC culture performed under standard Biological Safety 2 conditions as described (Ploner et al., and blastocyst injection; the Biological Resources Unit at Clare Hall 2008; Wolf et al., 2006). To generate the inducible FZR1 RNAi cell lines, TetR- and the ZVTA Innsbruck for animal husbandry; Christian Ploner for expressing cell lines were sequentially transduced with two shRNA-expressing lentiviral constructs conferring puromycin resistance (2.5 g/ml) and GFP expression, help with virus production; Reinhard Fässler (MPI Matrinsried respectively. To generate stable p53-knockdown cells, conditional FZR1 U2OS cell Germany) for Y2-865 cells; Jan-Michael Peters, Aloys Schepers and were transduced with a lentiviral shRNA vector coexpressing RFP (kindly provided Claudio Schneider for antibodies; and Elisabeth Sparber for excellent by Christian Ploner, Medical University of Innsbruck, Innsbruck, Austria). Infectious technical assistance. This work was supported by FWF grants SFB021 adenoviral particles were generated in 293A cells, purified using Vivapure Adenopack ‘Cell proliferation and cell death in tumors’, P16400, EU project LSHS- 20 (Sartorius, Goettingen, Germany) and titres determined by quantitative PCR (Ma et al., 2001) and correlation to Venus-positive cells by flow cytometry. For transient CT-2004-503438 ‘TRANSFOG’ as well as by the Tiroler Krebshilfe, RNAi, U2OS cells were transfected at 50% confluency with 60 nM siRNA targeting Tiroler Zukunftsstiftung and Tiroler Wissenschaftsfonds (TWF). FZR1 (5Ј-UGAGAAGUCUCCCAGUCAGTT-3Ј) or Luciferase (control) for 24 hours using Lipofectamine 2000 (Invitrogen). References Abramoff, M. D., Magelhaes, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Microscopy and image processing Biophotonics International 11, 36-42. Live-cell microscopy was performed on an Axiovert 200M microscope (Carl Zeiss, Almeida, A., Bolanos, J. P. and Moreno, S. (2005). Cdh1/Hct1-APC is essential for the Jena, Germany) as described (Wolf et al., 2006). Phase-contrast movies were survival of postmitotic neurons. J. Neurosci. 25, 8115-8121. obtained using a 10ϫ PLAN NeoFluar 0.3 NA objective with images taken every 5 Bartek, J., Bartkova, J. and Lukas, J. (2007). DNA damage signalling guards against minutes. Duration of mitosis was determined by counting the frames between cell activated oncogenes and tumour progression. Oncogene 26, 7773-7779. rounding and completion of cytokinesis by phase-contrast microscopy. Images and Bartkova, J., Rezaei, N., Liontos, M., Karakaidos, P., Kletsas, D., Issaeva, N., Vassiliou, movies were analysed using Metamorph 7.0 (Molecular Devices, Downingtown, PA), L. V., Kolettas, E., Niforou, K., Zoumpourlis, V. C. et al. (2006). Oncogene-induced converted to eight-bit TIFF format and further processed using Adobe Photoshop senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. CS2 and Adobe Illustrator CS2. Nature 444, 633-637. Bassermann, F., Frescas, D., Guardavaccaro, D., Busino, L., Peschiaroli, A. and Pagano, Gene targeting M. (2008). The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response Vector construction was carried out using a ~14 kb genomic FZR1 fragment cloned checkpoint. Cell 134, 256-267. from a -KO2 library (Zhang et al., 2002) by targeted insertion of a loxP-site-flanked Binne, U. K., Classon, M. K., Dick, F. A., Wei, W., Rape, M., Kaelin, W. G., Jr., Naar, TcR cassette into intron 8. After RS-recombinase-mediated release of the plasmid, A. M. and Dyson, N. J. (2007). Retinoblastoma protein and anaphase-promoting complex the TcR cassette was removed and a second loxP site introduced along with the physically interact and functionally cooperate during cell-cycle exit. Nat. Cell Biol. 9, eukaryotic NeoR selection marker into intron 2. The resulting conditional gene- 225-232. Journal of Cell Science targeting plasmid pKO2-FZR1-KO-II was partially sequenced, linearised using SalI Blanco, M. A., Sanchez-Diaz, A., de Prada, J. M. and Moreno, S. (2000). APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation. and used to electroporate R1 ES cells. G418-resistant cells were screened by Southern EMBO J. 19, 3945-3955. blotting and clones 3A3 and 4F1 used for blastocyst microinjection. After germline Brandeis, M. and Hunt, T. (1996). The proteolysis of mitotic cyclins in mammalian cells transmission, mice were backcrossed to C57Bl/6. Exon 2 to exon 8 of NeoR and persists from the end of mitosis until the onset of S phase. EMBO J. 15, 5280-5289. Fzr1 were sequentially deleted using transgenic Flpe and Cre mice, respectively. All Branzei, D. and Foiani, M. (2009). The checkpoint response to replication stress. DNA mouse breeding was performed according to the requirements of the animal protection Repair (Amst) 8, 1038-1046. act. Brummelkamp, T. R., Bernards, R. and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553. Immunoblotting, immunostaining, immunoprecipitation and kinase den Elzen, N. and Pines, J. (2001). Cyclin A is destroyed in prometaphase and can delay assay chromosome alignment and anaphase. J. Cell Biol. 153, 121-136. Immunoblotting and immunostaining were performed as described (Wolf et al., 2006). Di Micco, R., Fumagalli, M., Cicalese, A., Piccinin, S., Gasparini, P., Luise, C., Schurra, BrdU staining on fixed and acid-treated cells was done using the FLUOS in situ C., Garre’, M., Nuciforo, P. G., Bensimon, A. et al. (2006). Oncogene-induced proliferation kit (Roche Biochemicals, Vienna, Austria) and nuclei were counterstained senescence is a DNA damage response triggered by DNA hyper-replication. Nature 444, with 2 g/ml propidium iodide (PI). For CDC27 immunoprecipitation, AF3.1 638-642. antibodies were bound to Affiprep protein-A beads (Bio-Rad, Vienna, Austria) and Diffley, J. F. (2004). Regulation of early events in chromosome replication. Curr. Biol. 14, crosslinked using 40 mM dimethyl pimedilate in 0.1 M borate (pH 9). Control beads R778-R786. were prepared using 8C12 antibody (Wandke and Geley, 2006). MEFs were treated Engelbert, D., Schnerch, D., Baumgarten, A. and Wasch, R. (2008). The with 500 nM noc for 12 hours, harvested and released. The first time point was taken APC(Cdh1) is required to maintain genome integrity in primary human cells. Oncogene immediately after the first washing, whereas the remaining cells were plated at ~4ϫ106 27, 907-917. cells per 10-cm dish and incubated before being scraped off the plate in cold PBS Floyd, S., Pines, J. and Lindon, C. (2008). APC/C Cdh1 targets aurora kinase to control reorganization of the mitotic spindle at anaphase. Curr. Biol. 18, 1649-1658. for pelleting and lysis in 300 l of 20 mM HEPES-KOH pH 7.4, 150 mM NaCl,  Garcia-Higuera, I., Manchado, E., Dubus, P., Canamero, M., Mendez, J., Moreno, S. 0.1% Triton X-100, 1 mM EGTA, PhosphoStop (Roche), Complete (Roche) protease and Malumbres, M. (2008). Genomic stability and tumour suppression by the APC/C inhibitors and 50 g/ml PMSF for 60 minutes at 4°C. After centrifugation for 30 cofactor Cdh1. Nat. Cell. Biol. 10, 802-811. minutes, 350 g protein was mixed with 30 l equilibrated antibody beads for 1 hour, Geley, S., Kramer, E., Gieffers, C., Gannon, J., Peters, J. M. and Hunt, T. (2001). followed by three washes in lysis buffer, boiling in 20 l SDS-sample buffer and Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts analysis of 10 l by immunoblotting. at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell For immunoprecipitation of cyclin A, conditional FZR1 RNAi U2OS cells were Biol. 153, 137-148. treated for 3 days in the presence or absence of 1 g/ml dox before addition of 250 Hagting, A., den Elzen, N., Vodermaier, H. C., Waizenegger, I. C., Peters, J. M. and 6 nM noc for 12 hours. After mitotic shake-off, cells were replated at 4ϫ10 cell per Pines, J. (2002). Human securin proteolysis is controlled by the spindle checkpoint and 10 cm dish and harvested at 2, 4 and 6 hours, whereas the mitotic sample was taken reveals when the APC/C switches from activation by Cdc20 to Cdh1. J. Cell Biol. 157, immediately after the first wash. Lysis was carried out in 250 l lysis buffer (150 1125-1137. mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 0.1% NP40, Complete protease Jacobs, H., Richter, D., Venkatesh, T. and Lehner, C. (2002). Completion of mitosis inhibitors, PhosphoStop and 50 g/ml PMSF) for 1 hour on ice, before clearing for requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog. 30 minutes by centrifugation at 4°C. Protein (700 g) was incubated for 45 minutes Curr. Biol. 12, 1435-1441. FZR1 function in mitosis and G1 phase 4217

Kimata, Y., Baxter, J. E., Fry, A. M. and Yamano, H. (2008). A role for the Fizzy/Cdc20 Raff, J. W., Jeffers, K. and Huang, J. Y. (2002). The roles of Fzy/Cdc20 and Fzr/Cdh1 family of proteins in activation of the APC/C distinct from substrate recruitment. Mol. in regulating the destruction of cyclin B in space and time. J. Cell Biol. 157, 1139- Cell 32, 576-583. 1149. Kraft, C., Vodermaier, H. C., Maurer-Stroh, S., Eisenhaber, F. and Peters, J. M. (2005). Rape, M. and Kirschner, M. W. (2004). Autonomous regulation of the anaphase-promoting The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C complex couples mitosis to S-phase entry. Nature 432, 588-595. substrates. Mol. Cell 18, 543-553. Reis, T. and Edgar, B. A. (2004). Negative regulation of dE2F1 by cyclin-dependent kinases Kramer, E. R., Gieffers, C., Holzl, G., Hengstschlager, M. and Peters, J. M. (1998). controls cell cycle timing. Cell 117, 253-264. Activation of the human anaphase-promoting complex by proteins of the CDC20/Fizzy Schwab, M., Lutum, A. S. and Seufert, W. (1997). Yeast Hct1 is a regulator of Clb2 family. Curr. Biol. 8, 1207-1210. cyclin proteolysis. Cell 90, 683-693. Kramer, E. R., Scheuringer, N., Podtelejnikov, A. V., Mann, M. and Peters, J. M. (2000). Sivaprasad, U., Machida, Y. J. and Dutta, A. (2007). APC/C-the master controller of Mitotic regulation of the APC activator proteins CDC20 and CDH1. Mol. Biol. Cell 11, origin licensing? Cell Div. 2, 8. 1555-1569. Sudo, T., Ota, Y., Kotani, S., Nakao, M., Takami, Y., Takeda, S. and Saya, H. (2001). Li, M., Shin, Y. H., Hou, L., Huang, X., Wei, Z., Klann, E. and Zhang, P. (2008). The Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage- adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining induced G2 checkpoint in vertebrate cells. EMBO J. 20, 6499-6508. replicative lifespan and in learning and memory. Nat. Cell. Biol. 10, 1083-1089. Visintin, R., Prinz, S. and Amon, A. (1997). CDC20 and CDH1: a family of substrate- Ma, L., Bluyssen, H. A., De R. M., Laurysens, V., van der, B. N., Pavliska, H., van specific activators of APC-dependent proteolysis. Science 278, 460-463. Zonneveld, A. J., Tomme, P. and van Es, H. H. (2001). Rapid determination of Walker, A., Acquaviva, C., Matsusaka, T., Koop, L. and Pines, J. (2008). UbcH10 has adenoviral vector titers by quantitative real-time PCR. J. Virol. Methods 93, 181-188. a rate-limiting role in G1 phase but might not act in the spindle checkpoint or as part Mailand, N. and Diffley, J. F. (2005). CDKs promote DNA replication origin licensing in of an autonomous oscillator. J. Cell Sci. 121, 2319-2326. human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122, 915-926. Wandke, C. and Geley, S. (2006). Generation and characterization of an hKid specific Matyskiela, M. E. and Morgan, D. O. (2009). Analysis of activator-binding sites on the monoclonal antibody. Hybridoma 25, 41-43. APC/C supports a cooperative substrate-binding mechanism. Mol. Cell 34, 68-80. Wei, W., Ayad, N. G., Wan, Y., Zhang, G. J., Kirschner, M. W. and Kaelin, W. G., Jr. Nasmyth, K. (2002). Segregating sister genomes: the molecular biology of chromosome (2004). Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase- separation. Science 297, 559-565. promoting complex. Nature 428, 194-198. Pagano, M. (2004). Control of DNA synthesis and mitosis by the Skp2-p27-Cdk1/2 axis. Wirth, K. G., Ricci, R., Gimenez-Abian, J. F., Taghybeeglu, S., Kudo, N. R., Jochum, Mol. Cell 14, 414-416. W., Vasseur-Cognet, M. and Nasmyth, K. (2004). Loss of the anaphase-promoting Peters, J. M. (2006). The anaphase promoting complex/cyclosome: a machine designed complex in quiescent cells causes unscheduled hepatocyte proliferation. Genes Dev. 18, to destroy. Nat. Rev. Mol. Cell. Biol. 7, 644-656. 88-98. Pfleger, C. M. and Kirschner, M. W. (2000). The KEN box: an APC recognition signal Wolf, F., Wandke, C., Isenberg, N. and Geley, S. (2006). Dose-dependent effects of stable distinct from the D box targeted by Cdh1. Genes Dev. 14, 655-665. cyclin B1 on progression through mitosis in human cells. EMBO J. 25, 2802-2813. Pines, J. (2006). Mitosis: a matter of getting rid of the right protein at the right time. Trends Wolthuis, R., Clay-Farrace, L., van Zon, W., Yekezare, M., Koop, L., Ogink, J., Cell Biol. 16, 55-63. Medema, R. and Pines, J. (2008). Cdc20 and Cks direct the spindle checkpoint- Ploner, C., Rainer, J., Niederegger, H., Eduardoff, M., Villunger, A., Geley, S. and independent destruction of cyclin A. Mol. Cell 30, 290-302. Kofler, R. (2008). The BCL2 rheostat in glucocorticoid-induced apoptosis of acute Yu, H. (2007). Cdc20: a WD40 activator for a cell cycle degradation machine. Mol. Cell lymphoblastic leukemia. Leukemia 22, 370-377. 27, 3-16. Potapova, T. A., Daum, J. R., Pittman, B. D., Hudson, J. R., Jones, T. N., Satinover, Zhang, P., Li, M. Z. and Elledge, S. J. (2002). Towards genetic genome projects: genomic D. L., Stukenberg, P. T. and Gorbsky, G. J. (2006). The reversibility of mitotic exit library screening and gene- targeting vector construction in a single step. Nat. Genet. in vertebrate cells. Nature 440, 954-958. 30, 31-39. Journal of Cell Science