Mutual Antagonism Between the Anaphase Promoting Complex and the Spindle Assembly Checkpoint Contributes to Mitotic Timing in Caenorhabditis elegans

Abstract

The anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and exit from mitosis across eukaryotic evolution. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. Here, we report the characterization of a novel allele of the APC5 component SUCH-1 in Caenorhabditis elegans. We find that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/MAD1 or MDF-2/MAD2 rescues proper timing in these embryos. Moreover, partial depletion of the E1 enzyme uba-1 significantly increases mitosis duration upon monopolar spindle assembly. Taken together, our findings raise the possibility that the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos.

MITOSIS is tightly regulated in time and space to ensure equal distribution of sister chromatids and cytoplasmic constituents to daughter cells. Mistakes in chromosome segregation can cause aneuploidy and contribute to tumor progression. Therefore, a thorough knowledge of the mechanisms regulating mitosis is key for understanding proliferation control.

Bipolar attachment of sister chromatids to the mitotic spindle is crucial for their faithful segregation. Once bipolar attachment is achieved, the anaphase promoting complex/cyclosome (APC/C) triggers the metaphase–anaphase transition and mitotic exit. The APC/C is a multisubunit complex that functions as an E3 ligase, which ubiquitinates substrates, thereby marking them for destruction by the 26S proteasome (reviewed in Peters 2006). The recognition of substrates during mitosis is mediated primarily by the APC/C-associated coactivator Cdc20 (Visintin et al. 1997). Degradation of the APC/C substrates securin and cyclin B triggers the metaphase–anaphase transition and initiates mitotic exit, respectively (Murray and Kirschner 1989; Murray et al. 1989; Cohen-Fix et al. 1996). To ensure that cells do not enter anaphase until all chromosomes have achieved bipolar attachment, the spindle assembly checkpoint (SAC) monitors microtubule attachment and tension at kinetochores (Rieder et al. 1994; Li and Nicklas 1995). As long as the SAC is engaged, the SAC component MAD2 is present at kinetochores (Chen et al. 1996; Li and Benezra 1996), resulting in the sequestration of Cdc20 in a complex with MAD2 and/or BubR1 (Hwang et al. 1998; Kim et al. 1998; Tang et al. 2001; Nilsson et al. 2008). Therefore, the APC/C cannot efficiently ubiquitinate substrates, thus preventing their degradation and hence delaying progression through mitosis (Hwang et al. 1998; Kim et al. 1998). When complete bipolar chromosome attachment is achieved, MAD2 departs from kinetochores (Chen et al. 1996; Li and Benezra 1996), thus allowing Cdc20 to function as an APC/C coactivator and promote mitotic progression. Cells can also undergo what has been termed “mitotic slippage” and eventually progress through mitosis without satisfying the SAC (Rieder and Maiato 2004).

Despite the holocentric nature of Caenorhabditis elegans chromosomes (reviewed in Oegema and Hyman 2006), the SAC components and their function are conserved in nematodes. Thus, the checkpoint proteins MDF-1/MAD1 and MDF-2/MAD2 are required for viability and for germ cell arrest after nocodazole treatment (Kitagawa and Rose 1999). In early embryos, however, chemical or mutational disruption of the spindle does not result in arrest, but instead leads to a merely 1.5- to twofold delay during mitosis (Encalada et al. 2005; Essex et al. 2009). The mechanisms underlying such differential responses are only partially understood.

The relationship between the SAC and the APC/C is not fully elucidated either. Cdc20 is turned over in an APC/C-dependent manner during SAC engagement, both in budding yeast and in human cells (Pan and Chen 2004; Nilsson et al. 2008). It has thus been proposed that Cdc20 ubiquitination and degradation maintain SAC engagement in human cells (Nilsson et al. 2008; Ge et al. 2009). Accordingly, modest overexpression of Cdc20 inactivates the SAC in budding yeast, further indicating that low Cdc20 levels must be maintained for proper SAC function (Pan and Chen 2004). In apparent contradiction with this view, ubiquitination of Cdc20 is needed to inactivate the SAC in human cells (Reddy et al. 2007; Garnett et al. 2009). Overall, although the SAC and the APC/C have been studied extensively biochemically, their relationship remains incompletely understood, in particular in a living organism.

We have taken advantage of the early embryo of C. elegans to study aspects of the regulation of mitosis in vivo. Here, we report the phenotypic analysis of such-1(t1668), a mutation affecting an APC5 subunit. Our findings reveal that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle. Importantly, we found a substantial increase in the duration of mitosis in these embryos. Further analysis suggests that this results from prolonged engagement of the SAC. Our findings thus clarify the relationship between the SAC and the APC/C in a developing organism and establish that APC/C activity sets the duration of mitosis through the SAC in C. elegans embryos.

MATERIALS AND METHODS

Worm strains:

C. elegans strains of the following genotypes were cultured according to standard procedures (Brenner 1974): Bristol N2, Hawaiian CB4856, spd-5(or213) I (Hamill et al. 2002) (raised at 16° and shifted to 25° for 15 hr), smg-2(e2008) I (Hodgkin et al. 1989), emb-27(g48) II (raised at 16° and shifted to 22° for 15 hr) (Cassada et al. 1981), fzy-1(h1983) dpy-10(e128) II (raised at 16° and shifted to 25° for >15 hr) (Kitagawa et al. 2002), fzy-1(av15) unc-4(e120) II (Stein et al. 2007), and such-1(t1668) unc-32(e189) III/qCI dpy-19(e1259) glp-1(q339); him-3(e1147) IV (Gönczy et al. 1999) (raised at 15° or 16° unless otherwise indicated). plg-1(e2001) III males were used to unambiguously identify mated hermaphrodites owing to the presence of a gelatinous plug (Hodgkin and Doniach 1997). fog-2(q71) V females producing wild-type oocytes were used in crosses to determine the paternal contribution of such-1(t1668) (Schedl and Kimble 1988).

For deficiency mapping, we crossed such-1(t1668) unc-32(e189)/qCI hermaphrodites to tDf1/unc-32(e189) dpy-18(e499) III males. We selected wild-type F₁ animals and identified among the F₂′s hemizygous such-1(t1668) animals by the absence of Dpy or Dpy-Unc. Some F₁ animals were sterile, exhibiting an abnormal gonad and a protruding vulva.

For timing the metaphase–anaphase transition, we generated a strain carrying H2B–mCherry (McNally et al. 2006) in a such-1(t1668) unc-32(e189)/qCI background; embryos from homozygous such-1(t1668) hermaphrodites fertilized by plg-1(e2001) sperm with 100% bipolar spindles were analyzed. The strain such-1(h1960) III; unc-46(e177) mdf-1(gk2) V (Tarailo et al. 2007) was used to generate homozygous such-1(h1960) animals. For all experiments with such-1(h1960), L4 larva were shifted to 25° for 15 hr. We crossed homozygous such-1(h1960) males to fzy-1(av15) unc-4(e120) II (Stein et al. 2007) or MDF-2–GFP (Essex et al. 2009) hermaphrodites. The mutation in such-1(h1960) destroys an HpaII restriction site, which was followed in all crosses by PCR and RFLP. Transgenic lines were maintained at 24°.

In all experiments with such-1(t1668), L4 hermaphrodites were selected and kept separate at the indicated temperature to avoid fertilization by sibling males. For progeny tests, we quantified eggs laid after removal of the parents and hatched larva 24–48 hr later. We found that such-1(t1668) is 98.5% embryonic lethal at 15° and 100% at 25° (N > 1000 in each case). For simplicity, results from progeny tests carried out at 15°–25° were pooled. We found also that progeny from such-1(t1668) heterozygous mutant animals develop slightly slower than wild type, indicative of a semidominant effect on zygotic development.

For experiments involving mating, animals were kept at 20°. To address the paternal contribution to embryonic lethality, we generated the strain such-1(t1668)/qCI dpy-19(e1259) glp-1(q339). Single males from this strain were mated with several fog-2 females, embryonic lethality determined, and subsequently homozygous such-1(t1668) males identified by sequencing.

Matings for complementation analysis and deficiency mapping were carried out at 20° and animals then shifted to 25°. such-1(h1960) fails to complement such-1(t1668) for embryonic lethality; additionally, such-1(h1960)/such-1(t1668) embryos undergoing monopolar mitosis induced by spd-5(RNAi) are delayed during mitosis (43 min 10 sec ± 2 min 39 sec N = 7) like either homozygous mutant such-1 parental strain.

Mapping:

Unc-Emb and Unc-nonEmb recombinants were recovered from strains heterozygous for CB4856 and unc-32(e189) such-1(t1668). SNP mapping (Wicks et al. 2001) was then used to narrow the region between 10.75 cM and 11.51 cM with SNPs snp_Y66D12A[1] and haw47495. Sequencing of such-1 DNA from such-1(t1668) worms revealed a single nucleotide (A) deletion in exon 6 at position 1155. RT–PCR for such-1 was performed with gene-specific primers from nucleotide 1 until nucleotide 1461. In a second PCR step, this template was amplified with primers spanning exons 6 and 7 to reveal the ratio of the two mRNA species.

RNAi-mediated inactivation:

RNAi-mediated inactivation by feeding was performed by selecting L4 larva and treating them as follows: spd-5(RNAi) >15 hr at 25° (Rual et al. 2004), except for spd-5(RNAi) in emb-27(g48), which was carried out at 22°; mdf-1(RNAi) (from nucleotides 325 through 1522 of the gene amplified from genomic DNA and cloned in L4440) (Timmons and Fire 1998) and mdf-2(RNAi) (from nucleotides 75 through 575 of the gene amplified from genomic DNA and cloned in L4440) for 48 hr at 16°; and partial uba-1(RNAi) 13.5–19 hr at 25° (Rual et al. 2004), partial fzy-1(RNAi) 24 hr at 25° (Rual et al. 2004), and gfi-3(RNAi) >30 hr at 24° (Kamath et al. 2003).

We generated an RNAi feeding vector containing the first 1461 nucleotides of the such-1 cDNA cloned in L4440 (Timmons and Fire 1998). Feeding L1/L2 larva at 25° with such-1(RNAi) results in 100% embryonic lethality (N = 105) but no meiotic arrest. such-1(h1960) was reported to cause 46% embryonic lethality at 25° (Tarailo et al. 2007). In our hands, such-1(h1960) shifted as L4 is 10% and 90% embryonic lethal at 24° (N = 309) and 25° (N = 264), respectively. such-1(h1960) is not a paternal-effect allele, since the extent of embryonic lethality is not different among self-progeny and when such-1(h1960) are mated with plg-1(e2001) males (80% ± 3% SEM, N = 443 vs. 83% ± 3% SEM, N = 212) and since such-1(h1960) males do not cause significant embryonic lethality after fertilization of oocytes from fog-2(q71) females (3% lethality ± 6% SEM; N = 175). We investigated potential redundancy between such-1 and gfi-3; gfi-3(RNAi) alone does not affect viability but increases embryonic lethality in such-1(h1960) at 24° to 80% (N = 166).

Time-lapse microscopy:

Embryos were analyzed by time-lapse DIC microscopy on a Zeiss Axioskop 2 with ×100 A-plan objective (NA 1.25 oil) at 23° with a frame rate of 10 sec (Gönczy et al. 1999). Images were acquired with Scion Image and a Kappa CF 8/4 camera. M phase duration was measured from the first signs of nuclear envelope breakdown (NEBD; initial “shrinking” of the NE) until the first signs of nuclear envelope reformation (NER). Due to the inclusion of the first signs of NEBD and the fact that we did not use cytokinesis to time mitotic exit, M phase duration reported here for wild-type embryos is slightly longer than in the literature (Brauchle et al. 2003; Encalada et al. 2005; Hachet et al. 2007; Essex et al. 2009). Note that average times are given in the text in each case, with standard error of mean and number of embryos examined indicated in the corresponding figure panels. The calculated average duration was rounded up to the nearest 10 sec (except for movies with 30-sec time intervals, where the rounding up was to the nearest 30 sec). For spd-5 depletion, only embryos with a monopolar spindle were quantified.

Dual time-lapse fluorescence and DIC microscopy was carried out on a Zeiss Axioplan 2, ×63 Plan-Apochromat (NA 1.4 oil DIC) equipped with a RT monochrome spot camera 2.1.1 and Metamorph software (Sonneville and Gönczy 2004), with a frame rate of 10 sec, except such-1(h1960) spd-5(RNAi) embryos, which were imaged every 30 sec. The duration of MDF-2–GFP localization on chromosomes was quantified in wide-field recordings from the first signs of NEBD until the enrichment of the GFP signal on chromosomes disappeared.

For illustrating the MDF-2–GFP data (Figure 6), embryos were imaged on an LSM700 Zeiss confocal microscope with a ×40 Plan-Apochromat (NA 1.3 oil) objective, capturing a single optical slice of 3 μm at a time interval of 10 sec for spd-5(RNAi) and of 10 sec (early mitosis) up to 10 min (during the prolonged mitosis) for such-1(h1960) spd-5(RNAi) embryos.

Figure 6.—

The substantial increase in M phase duration in APC/C mutants with monopolar mitosis is caused by SAC engagement. (A) MDF-2–GFP localization in one-cell–stage embryos of the indicated genotypes. Note that MDF-2–GFP accumulates for an extended period on chromatin of such-1(t1668) spd-5(RNAi) embryos. Time in min:sec relative to onset of NEBD. Arrowhead points to MDF-2–GFP enrichment on chromatin. (B) Duration of MDF-2–GFP enrichment on chromatin measured from NEBD. (C) Inactivation of the SAC through mdf-1 or mdf-2 depletion reduces M phase delay in hypomorphic APC/C mutants.

Indirect immunofluorescence:

Fixation and indirect immunofluorescence were performed essentially as described (Gönczy et al. 1999). The following primary antibodies were used: mouse α-tubulin (DM1A, Sigma; 1/300), mouse SP-56 (a generous gift from Susan Strome (Ward et al. 1986; 1/100), rabbit SAS-4 (Leidel and Gönczy 2003; 1/600), and rabbit TAC-1 (Bellanger and Gönczy 2003; 1/300). Secondary antibodies were goat anti-mouse coupled to Alexa 488 (1/1000) and goat anti-rabbit coupled to Alexa 568 (1/1000) (Molecular Probes). DNA was revealed with ∼1 μg/ml Hoechst 33258 (Sigma). Images were acquired on a LSM 700 Zeiss (embryos) or SP2 Leica (sperm) confocal microscope. Maximal intensity projections of selected confocal planes are shown and were generated in ImageJ.

RESULTS

Aberrant centrosome number and DNA content in such-1(t1668) embryos:

Mitosis can be analyzed with high temporal and spatial resolution in one-cell–stage C. elegans embryos using time-lapse DIC microscopy. In the wild-type (supporting information, File S1), there is one male and one female pronucleus (Figure 1A, −5:00). The female pronucleus forms at the presumptive anterior and then moves toward the male pronucleus, which is located at the posterior, associated with the centrosomes. The two joined pronuclei and the accompanying centrosomes then move to the cell center. The two centrosomes serve as microtubule-organizing centers (MTOCs), which organize bipolar spindle assembly following NEBD, thus ensuring faithful chromosome segregation (Figure 1A, +2:30; Figure 2A). Cytokinesis and NER mark the end of mitosis, resulting in the generation of two daughter cells, each with a full complement of chromosomes (Figure 1A, +9:00).

Figure 1.—

Phenotypes of such-1(t1668) embryos: still images from time-lapse DIC recordings; time relative to onset of NEBD in min:sec. Anterior or polar body position is left in this and other figures. Bar, 10 μm. Asterisks, pronuclei (female left, male right); black arrowheads, spindle poles; white arrowhead, center of monopolar configuration. See also File S1, File S2, File S3, File S4. (A) (−5:00): wild-type embryo with one female and one male pronucleus; a pseudocleavage furrow forms. (+2:30) A bipolar spindle assembles during mitosis. (+9:00) The first division gives rise to a larger anterior AB cell and a smaller posterior P₁ cell. (B–D) such-1(t1668) embryos classified according to the number of pronuclei and the presence of centrosomes. All embryos examined contain a single female pronucleus with normal size. N = 38; percentages on the right indicate the fraction of embryos within each class. (B) (−5:00) Class I embryos possess one male and one female pronucleus; a pseudocleavage furrow forms. (+2:30) These embryos assemble a spindle that is bipolar (5/11) or multipolar (6/11; not shown, presumably when supernumerary centrioles were contributed by the sperm). (+9:00) All class I embryos divide. (C) (−5:00) Class II embryos have a female pronucleus, but no male pronucleus; pseudocleavage occurs. (+2:30) Centrosomes are present and form either a bipolar spindle (10/21) or a multipolar spindle (6/21; not shown). Sometimes the spindle appears monopolar initially, with strong microtubule nucleation from the MTOC, but resolves within the same prolonged cell cycle into a bipolar spindle (5/21; not shown), possibly reflecting a defect in the timing of centriole disengagement. (+9:00) All class II embryos divide. (D) (−5:00) Class III embryos have a female pronucleus, but no male pronucleus; no pseudocleavage occurs. (+2:30) MTOCs are absent and embryos undergo monopolar mitosis, with microtubules nucleated weakly around the chromatin (6/6; see also Figure 2C). (+9:00) Embryos are still in mitosis and fail to divide even after NER and exit from mitosis (see File S4).

Figure 2.—

such-1(t1668) embryos lacking centrioles undergo monopolar mitosis: immunofluorescence of mitotic one-cell–stage embryos of the indicated parental genotypes. Centrioles (SAS-4, red), microtubules (α-tubulin, green), and Hoechst counterstain to visualize DNA (blue). Insets show sixfold-magnified view of the SAS-4 signal in the center of the asters. Bar, 10 μm. (A) Wild-type embryo with four centrioles; the two centrosomes nucleate two dense microtubule asters and direct bipolar spindle assembly. (B) such-1(t1668) class I or II embryo (the two classes cannot be distinguished in fixed specimens during mitosis) with four centrioles and a bipolar spindle. A total of 1/28 embryos with a monopolar spindle harbored centrioles and a dense array of microtubules, probably corresponding to the subset of embryos with a putative defect in the timing of centriole disengagement (described in the legend of Figure 1C). (C) such-1(t1668) class III embryo, which lack centrioles. Microtubules are nucleated around the chromatin, resulting in monopolar mitosis. (D) spd-5(or213) embryo, in which centrosome function is compromised despite the presence of centrioles, thus also resulting in monopolar mitosis.

such-1(t1668) is a recessive mutant allele identified in a collection of EMS-induced parental effect lethal mutations (Gönczy et al. 1999). The number of eggs laid by such-1(t1668) homozygous mutant animals is on average 92 ± 5 SEM (N = 17 adults), compared to 255 ± 13 SEM (N = 9 adults) for heterozygous siblings. We conducted time-lapse DIC microscopy to investigate the nature of the defect in embryos from homozygous such-1(t1668) hermaphrodites (hereafter referred to as such-1(t1668) embryos for simplicity). This enabled us to define three classes of such-1(t1668) embryos (N = 38 embryos). Class I embryos (File S2; 29%) have one male and one female pronucleus and assemble a bipolar spindle (Figure 1B; Figure 2B). Some class I embryos harbor more than two MTOCs and consequently assemble a multipolar spindle (data not shown; legend of Figure 1). Class II embryos (File S3; 55%) possess a female pronucleus, but no male pronucleus, and are thus haploid (Figure 1C, −5:00). Nevertheless, MTOCs are present at the embryo posterior and the female pronucleus migrates toward them as in the wild type. The single pronucleus and associated centrosomes then move to the cell center and during mitosis the two MTOCs assemble a bipolar spindle (Figure 1C, +2:30). Occasional class II embryos have more than two MTOCs and assemble a multipolar spindle (data not shown; legend of Figure 1). Most interestingly, class III embryos (File S4; 16%) also have a female pronucleus but no male pronucleus and are thus haploid (Figure 1D, −5:00). Here, however, no MTOC is apparent and the female pronucleus drifts slowly toward the cell center. Bipolar spindle assembly does not occur during mitosis. Instead, a monopolar configuration is observed, which will be referred to as monopolar mitosis hereafter (Figure 1D, +2:30; Figure 2C). This phenotype is reminiscent of that observed in spd-5 mutant embryos, which cannot nucleate robust microtubules from their defective centrosomes (Hamill et al. 2002). Similarly, we found that microtubules appear to be nucleated around the chromatin in class III such-1(t1668) embryos (Figure 2C). Class III embryos fail to divide after M phase exit (File S4).

To further investigate the apparent lack of centrosomes in class III embryos, we addressed whether these embryos contain centrioles using antibodies against the centriolar protein SAS-4 (Kirkham et al. 2003; Leidel and Gönczy 2003). In the wild type, each spindle pole contains two juxtaposed centrioles (Figure 2A). Classes I and II such-1(t1668) embryos are indistinguishable from the wild type with respect to SAS-4 distribution (Figure 2B). By contrast, SAS-4 foci are invariably absent in class III such-1(t1668) embryos (Figure 2C; 27/27 embryos). Accordingly, foci of the centrosomal protein TAC-1 are also absent from class III embryos (Figure S1C).

Taken together, these findings establish that some such-1(t1668) embryos lack a male pronucleus as well as centrioles and undergo monopolar mitosis. Furthermore, these results demonstrate that centrioles are dispensable for fertilization.

such-1(t1668) sperm has aberrant centriole numbers and missing DNA:

Since sperm contributes the DNA making up the male pronucleus as well as the sole centrioles to the zygote, we addressed whether some such-1(t1668) sperm exhibit abnormal DNA contents and centriole numbers.

Wild-type sperm contain highly condensed DNA associated with a pair of centrioles marked by SAS-4 (Figure 3A). These two centrioles usually appear as a single focus, due to their small size and proximity, which is usually below the resolution limit of the light microscope. such-1(t1668) sperm display an array of phenotypes with respect to DNA contents and centriole number that we classified by analogy with the embryonic phenotypes (N = 87 sperm). Class I sperm (32%) contain DNA and one or two separate SAS-4 foci, presumably corresponding to two or four centrioles (Figure 3B; data not shown). This subset of mutant sperm probably results in class I embryos, which are diploid and assemble a bipolar or a multipolar spindle. In class II sperm (47%), the DNA is absent, and one focus of SAS-4 is usually present (Figure 3C). This subset of such-1(t1668) sperm presumably results in class II haploid embryos lacking solely the male pronucleus. Class III sperm (21%) lack DNA, as well as centrioles (Figure 3D), and thus likely give rise to class III such-1(t1668) embryos.

Figure 3.—

such-1(t1668) sperm with aberrant DNA and centriole content causes embryonic lethality. (A–D) Immunofluorescence of wild-type and such-1(t1668) sperm. Centrioles (SAS-4, red), sperm membranes (SP-56, green), and Hoechst counterstain to visualize DNA (blue). Bar, 1 μm. Wild type, N = 58; such-1(t1668), N = 87; percentages on the right indicate the fraction of such-1(t1668) sperm of each class. Sperm with abnormal shapes or decondensed DNA were not considered. (A) Wild-type sperm with highly condensed DNA and one clear SAS-4 focus (corresponding to a pair of tightly apposed centrioles). (B) such-1(t1668) class I sperm with DNA. These sperm contain a single SAS-4 focus (6/28, corresponding to a pair of tightly apposed centrioles), two SAS-4 foci (19/28, presumably corresponding to four centrioles; not shown), or no visible SAS-4 (3/28, not shown). (C) such-1(t1668) class II sperm without DNA but with centrioles. These sperm contain either a single SAS-4 focus (35/41) or two SAS-4 foci (6/41, not shown). (D) such-1(t1668) class III sperm without DNA or centrioles (18/18); the cell was identified as sperm, based on its shape, size, and SP-56 staining. (E) such-1(t1668) is paternal effect embryonic lethal. Average embryonic lethality of progeny from wild type or such-1(t1668) hermaphrodites, such-1(t1668) hermaphrodites mated with plg-1(e2001) males and exhibited a gelatinous plug on the vulva, as well as fog-2(q71) females mated by such-1(t1668) males. Error bars, standard error of mean; N = number of embryos from a minimum of 21 hermaphrodites or four such-1(t1668) males.

Given the suggestive parallel between the defects in such-1(t1668) sperm and embryos, as well as the related proportions of each phenotypic class, we addressed whether the observed embryonic lethality is of paternal origin. In homozygous such-1(t1668) self-fertilizing hermaphrodites, mutant oocytes are fertilized by mutant sperm, yielding 100% embryonic lethality (Figure 3E). By contrast, we found that embryonic lethality is rescued when such-1(t1668) mutant oocytes are fertilized with wild-type sperm contributed by plg-1(e2001) males (Figure 3E). Moreover, such-1(t1668) males crossed to spermless fog-2(q71) females cause 100% embryonic lethality in the offspring (Figure 3E). We conclude that such-1(t1668) sperm causes paternal-effect embryonic lethality.

While the missegregation of DNA and centrioles likely occurs during sperm meiosis, the rescue of viability by wild-type sperm indicates that both meiotic divisions in such-1(t1668) oocytes occur normally. Despite the full rescue by wild-type sperm, the duration of mitosis in the resulting embryos is on average 33% longer than in the wild type (described below), a slight extension, which is tolerated by the developing organism (see also Tarailo et al. 2007).

Molecular lesion in such-1(t1668):

To identify the molecular nature of the such-1(t1668) allele, we initially carried out deficiency mapping. We found that the deficiency tDf1 fails to complement such-1(t1668) and that such-1(t1668)/tDf1 animals have an abnormal gonad and are almost sterile, precluding analysis of the resulting embryos. Additionally, some hemizygous animals have a protruding vulva, indicating that SUCH-1 has additional functions later during development. We conclude that such-1(t1668) is a reduction-of-function allele of a gene located on the right arm of chromosome III. Further SNP mapping placed such-1(t1668) within a 119-kbp interval between 10.77 cM and 11.51 cM (Figure 4A). Of 20 predicted genes within this region, such-1 was a plausible candidate since it encodes an APC5 protein, which is a subunit of the APC/C (Tarailo et al. 2007). Indeed, some hypomorphic APC/C alleles exhibit phenotypes partly reminiscent of the ones reported here (Golden et al. 2000; Sadler and Shakes 2000). We found that animals transheterozygous between such-1(t1668) and the previously identified allele such-1(h1960) give rise to 91% (±3% SEM, N = 1068) embryonic lethality, whereas the progeny of such-1(t1668) or such-1(h1960) heterozygous animals is viable (data not shown; Tarailo et al. 2007). Thus, such-1(h1960) fails to complement such-1(t1668) in terms of embryonic lethality.

Figure 4.—

Molecular characterization of such-1(t1668). (A) Mapping of such-1(t1668): SNP mapping positioned such-1(t1668) between 10.77 cM and 11.51 cM on the right arm of LGIII. The such-1 mRNA contains 12 exons. (B) Schematic representation of exons 6 and 7 and of the intervening intron 6 in wild-type and such-1(t1668) animals, as well as image of relevant RT–PCR products. In the wild type, the most prevalent mRNA species is one in which intron 6 is spliced out (lane 1, bottom band, small mRNA). By contrast, in such-1(t1668), the most prevalent mRNA species retains intron 6 (lane 2, top band, large mRNA). This change in ratio is due to the fact that the small mRNA is degraded in such-1(t1668) embryos by the NMD pathway, since the small mRNA prevails in such-1(t1668) smg-2(e2008) animals (lane 4).

Sequencing of such-1 in such-1(t1668) revealed a single nucleotide deletion in exon 6, which causes a frameshift creating a premature stop codon in exon 7 (Figure 4B, schematic). Since transcripts with premature stop codons are targeted for degradation by nonsense-mediated decay (NMD) (Pulak and Anderson 1993), such-1 mRNA from such-1(t1668) should be significantly diminished. To test this prediction, we amplified exons 6–7 from wild-type and such-1(t1668) mutant animals using RT–PCR. As shown in Figure 4B, wild-type animals yield a clear amplification product corresponding to exons 6–7, without the intervening intron (lane 1, bottom). This amplification product is significantly diminished in such-1(t1668) animals (lane 2, bottom). Inhibition of NMD using a mutation in smg-2 restores the levels of this amplification product (lane 4, bottom), confirming that the such-1(t1668) transcript containing a stop codon is usually degraded by NMD. such-1(t1668) smg-2(e2008) animals give rise to an embryonic lethality of 59% ± 13% SEM (N = 566) at 20°, compared to 100% for such-1(t1668) (see Figure 3E). Thus, increasing mutant SUCH-1 levels rescue viability, indicating that the protein is at least partially functional.

Furthermore, RT–PCR analysis and sequencing revealed that such-1(t1668) animals accumulate a longer transcript that retains the intron between exons 6 and 7 (Figure 4B, schematic and lane 2, top). This retention corrects the frameshift from exon 7 onward, such that this transcript is prevalent in such-1(t1668). Trace amounts of this longer transcript are also present in the wild type (Figure 4B, lane 1, top). The longer transcript in such-1(t1668) is predicted to yield a protein in which 21 amino acids encoded by exon 6 are altered due to the frameshift, and 20 extraneous amino acids are encoded by the intron. However, the N and C termini of SUCH-1 are unchanged, perhaps accounting for the residual function in such-1(t1668) animals.

Prolonged M phase in such-1(t1668) embryos with monopolar mitosis:

Further analysis of such-1(t1668) embryos shed new light on the mechanisms regulating mitosis in developing C. elegans embryos.

In wild-type one-cell–stage embryos, the time from the beginning of NEBD until NER is on average 5 min 30 sec, as measured by DIC time-lapse microscopy (Figure 5A). Previous work established that severe loss of APC/C function provokes metaphase arrest in meiosis I, whereas partial reduction of APC/C function does not prevent the execution of meiosis but delays the metaphase–anaphase transition and exit from mitosis in one-cell–stage embryos (Furuta et al. 2000; Golden et al. 2000; Shakes et al. 2003; Tarailo et al. 2007; McCarthy Campbell et al. 2009). To address whether there is a delay in the metaphase–anaphase transition in such-1(t1668) embryos, we analyzed embryos derived from mutant oocytes fertilized by wild-type sperm, which all set up a bipolar spindle. Using mCherry–H2B (McNally et al. 2006) to monitor chromosomes, we found that the time separating NEBD from the metaphase–anaphase transition is 2 min 20 sec on average in the wild type (±5 sec SEM, N = 10). By contrast, this increases to 3 min 40 sec in the such-1(t1668) background (±16 sec SEM, N = 9). Thus, the metaphase–anaphase transition is slightly delayed in such-1(t1668) embryos with a bipolar spindle, as anticipated for a hypomorphic allele of an APC/C component.

Figure 5.—

Monopolar mitosis in APC/C mutants substantially increases M phase duration. Bars represent average M phase duration in one-cell–stage embryos of the indicated genotypes measured from the beginning of NEBD until NER in DIC time-lapse recordings. N = number of embryos. Error bars, standard error of mean. (A) such-1(t1668) class I and class II embryos with a bipolar spindle are delayed only slightly during M phase, while class III embryos are delayed sevenfold. (B) Monopolar mitosis in APC/C mutants substantially increases M phase duration. Note that all mutant embryos are delayed upon spd-5(RNAi). (C) Ubiquitination influences M phase duration in monopolar mitosis. Albeit partial RNAi of uba-1 [in addition to spd-5(or213)] provokes a modest increase in M phase duration, the difference with partial uba-1(RNAi) alone is statistically significant (paired Student's T-test; P = 1.9 × 10⁻⁵). (D) Partial reduction of FZY-1 function by partial RNAi or with fzy-1(h1983) provokes a modest but statistically significant increase in M phase duration in embryos with a monopolar spindle induced by SPD-5 depletion (paired Student's T-test compared to fzy-1(RNAi) P = 1.4 × 10⁻⁴; fzy-1(h1983) P = 3.7 × 10⁻⁵). fzy-1(av15) rescues M phase duration in conditions that normally cause a drastic delay (compare to B).

As predicted also, we found that all such-1(t1668) embryos exhibit prolonged M phase duration. Importantly in addition, we found that the extent of this prolongation depends on the phenotypic class of such-1(t1668) embryos. Since anaphase cannot be scored in embryos with a monopolar spindle, the duration of mitosis in the following sections refers to the time separating NEBD from NER in DIC time-lapse recordings. As reported in Figure 5A, the duration of mitosis is prolonged only slightly in class I and class II embryos that assemble a bipolar spindle (6 min 50 sec and 7 min 20 sec, respectively), which corresponds to a 1.3-fold delay compared to the wild type. Strikingly, M phase lasts 39 min 10 sec in class III such-1(t1668) embryos that undergo monopolar mitosis, corresponding to a sevenfold increase compared to the wild type (Figure 5A). By contrast to the effects on mitosis, the duration of interphase is similar to the wild type in all three classes of such-1(t1668) embryos (Figure S2). Overall, we conclude that class III such-1(t1668) embryos are compromised in their capacity to exit mitosis.

Prolonged M phase in APC/C mutants with monopolar mitosis:

We set out to investigate the mechanisms leading to the substantial prolongation of mitosis in class III such-1(t1668) embryos. To address whether it is the lack of bipolar spindle assembly in class III such-1(t1668) embryos that causes this prolongation, we sought to prevent bipolar spindle assembly also in class I and class II embryos. To this end, we depleted in addition SPD-5, a coiled-coil protein required for centrosome assembly and hence bipolar spindle formation (Hamill et al. 2002). Embryos lacking SPD-5 function, obtained through RNAi-mediated depletion or using the mutant spd-5(or213), enter mitosis with two nonfunctional centrosomes. In the absence of MTOCs, microtubules are nucleated instead around the chromatin, thus mimicking the monopolar configuration of such-1(t1668) class III embryos (Figure 2D; File S5, compare to Figure 2C and File S4). The duration of M phase in spd-5(RNAi) embryos is slightly increased compared to the wild type, to 6 min 10 sec (Figure 5B). Importantly, we found that such-1(t1668) spd-5(RNAi) embryos all exhibit an increase in M phase duration to 41 min 30 sec (Figure 5B). We conclude that monopolar mitosis provokes a substantial prolongation of mitosis in such-1(t1668) embryos. To test whether the delay is of maternal origin, we timed M phase duration in such-1(t1668) spd-5(RNAi) embryos fertilized by wild-type sperm. In this case, monopolar mitosis lasts 37 min 50 sec ± 6 min 13 sec (N = 12), similar to the timing in self-fertilized embryos. Thus, the prolonged M phase upon monopolar mitosis is of maternal origin.

Next, we addressed whether prolonged M phase duration in such-1(t1668) embryos upon monopolar mitosis is allele specific. We analyzed such-1(h1960), another reduction-of-function allele that exhibits a minor increase in M phase duration upon bipolar spindle assembly (7 min; Figure 5B) (Tarailo et al. 2007). Strikingly, we found that such-1(h1960) spd-5(RNAi) embryos, which all undergo monopolar mitosis, spend 49 min 10 sec in mitosis (Figure 5B). Therefore, increased M phase duration upon monopolar mitosis is a common consequence of compromising SUCH-1 function.

Since SUCH-1 is an APC/C component, we addressed whether prolonged M phase is a general response to monopolar mitosis among hypomorphic APC/C mutants. To this end, we used emb-27(g48), a temperature-sensitive allele of the APC/C subunit APC6 (Cassada et al. 1981; Golden et al. 2000). emb-27(g48) embryos all arrest in metaphase of meiosis I at the restrictive temperature of 25° (Cassada et al. 1981; Golden et al. 2000). To circumvent this arrest, we used the semipermissive temperature of 22°, which enables progression through meiosis and mitosis and results in 10% embryonic lethality (N = 80). As expected from a reduction of APC/C function, the duration of mitosis in such embryos with a bipolar spindle is slightly increased, to 6 min 10 sec (Figure 5B). emb-27(g48) spd-5(RNAi) embryos instead assemble a monopolar spindle and remain in M phase for 48 min 40 sec (Figure 5B). Taken together, these findings reveal that, whereas reduction of APC/C function alone causes merely a minor prolongation of M phase, assembling a monopolar spindle under such conditions results in a drastic increase in the duration of mitosis.

Ubiquitination influences M phase duration upon monopolar mitosis:

The APC/C is an E3 ligase, which functions at the terminal step of a cascade forming ubiquitin chains on substrates (reviewed in Peters 2006). Prior to that step, ubiquitin is activated by an E1 enzyme and transferred to an E2 enzyme, which cooperates with the E3 ligase to ubiquinate substrates. We set out to test whether increased M phase duration upon monopolar mitosis is provoked by insufficient ubiquitination of APC/C substrates. If this were the case, then interfering with the ubiquitination cascade should also prolong the duration of monopolar mitosis. To test this prediction, we depleted by RNAi uba-1, the sole gene encoding an E1-activating enzyme in C. elegans (Kulkarni and Smith 2008). Due to its widespread requirement, strong RNAi-mediated inactivation of uba-1 causes sterility and metaphase arrest during meiosis I (Maeda et al. 2001; Jones et al. 2002; Sonnichsen et al. 2005). To allow entry into mitosis, we used partial inactivation conditions, which results in M phase lasting 6 min 30 sec upon bipolar spindle assembly (Figure 5C). Importantly, we found that the duration of M phase in uba-1(RNAi) spd-5(or213) embryos, which all undergo monopolar mitosis, is increased to 14 min 50 sec (Figure 5C). Overall, we conclude that ubiquitination by the APC/C participates in setting the duration of M phase upon monopolar mitosis.

The prolonged M phase is likely transmitted via the APC/C coactivator FZY-1/Cdc20:

We then asked how the APC/C could provoke such a delay upon monopolar mitosis. Since the APC/C functions with its coactivator FZY-1/Cdc20 during mitosis, we addressed whether FZY-1/Cdc20 participates in prolonging M phase in APC/C mutants in a monopolar setting. As for other APC/C components and UBA-1, strong RNAi-mediated inactivation of FZY-1 causes metaphase arrest in meiosis I (Kitagawa et al. 2002). Therefore, we again used partial RNAi-mediated inactivation, which results in mitosis lasting 7 min 40 sec upon bipolar spindle assembly (Figure 5D). When FZY-1 was inactivated in this manner in spd-5(or213) embryos, the duration of mitosis is lengthened to 10 min 10 sec (Figure 5D). Similarly, whereas M phase lasts 8 min 10 sec in the weak reduction-of-function allele fzy-1(h1983), it lasts 10 min 30 sec in fzy-1(h1983) spd-5(RNAi) embryos (Figure 5D). These slight prolongations upon FZY-1 partial inactivation are statistically significant (legend of Figure 5) and indicate that prolonged M phase duration is at least partially dependent on FZY-1.

As a further test of the requirement for FZY-1 function in this delay, we used the mutant allele fzy-1(av15), which has been isolated as a suppressor of the hypomorphic APC/C mutant mat-3(or180) (Stein et al. 2007). It has been proposed that this mutation may interfere with MDF-2 binding, thus mimicking the loss of SAC signaling (Stein et al. 2007). If FZY-1 is required for prolonged M phase during monopolar mitosis, then fzy-1(av15) might be expected to alleviate this effect. As shown above, such-1(h1960) spd-5(RNAi) one-cell–stage embryos spend 49 min 10 sec in mitosis (Figure 5B). By contrast, such-1(h1960) fzy-1(av15) spd-5(RNAi) embryos undergoing monopolar mitosis spend only 8 min 20 sec in M phase (Figure 5D). This finding further supports the notion that FZY-1/Cdc20 is involved in prolonging M phase duration in APC/C mutants.

Prolonged M phase in APC/C mutants results from excess activation of the spindle assembly checkpoint:

Next, we investigated whether the substantial increase in M phase duration in such-1 embryos upon monopolar mitosis is caused by SAC engagement. This is of particular interest because the slight delay observed in such-1(h1960) embryos that assemble a bipolar spindle is independent of the SAC (Tarailo et al. 2007). As in all eukaryotes, the MAD2 checkpoint protein MDF-2 accumulates on unattached kinetochores in C. elegans and can be used as readout for SAC activation (Chen et al. 1996; Li and Benezra 1996; Kitagawa and Rose 1999; Essex et al. 2009). In wild-type one-cell–stage embryos, MDF-2–GFP enters the nucleoplasm at prometaphase and remains present as a diffuse cloud until the onset of anaphase (Figure 6A; Essex et al. 2009). In line with previous observations in embryos undergoing monopolar spindle assembly, we found that MDF-2–GFP is detected on chromatin during 2 min 10 sec on average in spd-5(RNAi) embryos (Figure 6, A and B) (Essex et al. 2009). Strikingly, we found that MDF-2–GFP chromatin localization is prolonged to 29 min in such-1(h1960) spd-5(RNAi) embryos (Figure 6, A and B), which corresponds to a 13-fold increase. Thus, prolonged M phase duration in APC/C mutants upon monopolar mitosis correlates with significant persistence of MDF-2–GFP on chromatin.

To test whether it is indeed SAC engagement that provokes prolonged M phase duration in APC/C mutants upon monopolar mitosis, we depleted the C. elegans checkpoint components MDF-1 (a homolog of MAD1) or MDF-2 (Kitagawa and Rose 1999). Importantly, we found that the duration of monopolar mitosis in such-1(h1960) mdf-1(gk2) spd-5(RNAi) is reduced to 10 min (Figure 6C). Similarly, mdf-1(RNAi) or mdf-2(RNAi) rescues the prolongation of M phase in such-1(t1668) class III embryos to 9 min 50 sec and 10 min, respectively (Figure 6C). The slight remaining prolongation compared to spd-5(RNAi) embryos may reflect an MDF-1/MDF-2 independent delay, as observed previously in other contexts in worms, flies, and humans (Orr et al. 2007; Tarailo et al. 2007; Williamson et al. 2009).

DISCUSSION

Aberrant sperm, spindle assembly, and centriole number:

We found that such-1(t1668) sperm contains aberrant DNA contents and centriole numbers. These phenotypes are partly reminiscent of the absence of paternal DNA and the presence of additional centrioles observed in some alleles of emb-27, emb-30, mat-1, mat-2, and mat-3, which all encode homologs of APC/C subunits (Furuta et al. 2000; Golden et al. 2000; Sadler and Shakes 2000). Given that the embryonic lethality of such-1(t1668) embryos is of paternal origin, the sperm defects fully explain the range of phenotypes observed in the resulting embryos. Thus, like other APC/C components, the APC5 subunit SUCH-1 is important for faithful segregation of DNA, centrioles, and perhaps other organelles during the meiotic divisions of male germ cells. In contrast, the female meiotic divisions seem normal in such-1(t1668) mutant animals, given notably that wild-type sperm is sufficient to rescue lethality. Thus, even though the analysis of animals with more complete inactivation of function may alter this conclusion, it appears that SUCH-1 has different requirements in the male and the female germline.

C. elegans mutant embryos that possess centrioles but cannot nucleate microtubules from their defective centrosomes do not assemble a bipolar spindle (Hamill et al. 2002). Here, we establish that the same is true for embryos that lack centrioles altogether. In other systems, including Xenopus egg extracts, Drosophila neuroblasts and vertebrate cells, a bipolar spindle can assemble in the absence of centrosomes owing to nucleation of microtubules around chromatin and their subsequent organization into a bipolar array (Heald et al. 1996; Khodjakov et al. 2000; Basto et al. 2006). This mechanism is critical for assembling the acentrosomal meiotic spindle, but can also be active in cells with centrosomes (Heald et al. 1997; Khodjakov et al. 2000). In early C. elegans embryos, by contrast, although some microtubules are nucleated in the vicinity of chromatin, they do not get organized into a bipolar array. Thus, even though the acentrosomal pathway of spindle assembly is active during the female meiotic divisions in C. elegans, it is either absent or inefficient during the mitotic division that follows.

Many cells possess the capacity to form centrioles de novo, for instance following ablation of resident centrioles in human cells or in parthenogenetic-activated oocytes (Miki-Noumura 1977; Szollosi and Ozil 1991; Khodjakov et al. 2002). Our analysis indicates that this is not the case in early C. elegans embryos, at least during the first two cell cycles, since we did not observe de novo centriole formation by time-lapse DIC microscopy among class III embryos.

APC5-related subunits in C. elegans:

Across eukaryotic evolution, the complete loss of APC/C function blocks the cell cycle at the metaphase–anaphase transition. Accordingly, newly fertilized C. elegans embryos lacking APC/C function arrest during metaphase of meiosis I (Furuta et al. 2000; Golden et al. 2000; Davis et al. 2002). By contrast, both meiotic divisions are completed in embryos of the two extant such-1 alleles. However, neither such-1(t1668) nor such-1(h1960) is a molecular null, and the partial rescue of embryonic viability in such-1(t1668) smg-2(e2008) animals suggests that the corresponding mutant protein retains residual activity. More importantly, there are two genes encoding APC5 proteins in C. elegans, such-1 and gfi-3 (Davis et al. 2002; Tarailo et al. 2007). Both corresponding transcripts are expressed in the gonad and the embryo (Y. Kohara, personal communication; http://nematode.lab.nig.ac.jp). Although gfi-3(RNAi) increases the embryonic lethality conferred by such-1(h1960) animals, it does not confer meiotic arrest (data not shown). However, subjecting such-1 mutant animals simultaneously to gfi-3(RNAi) and such-1(RNAi) results in the vast majority of embryos being arrested in meiosis I (Stein et al. 2010, accompanying article in this issue), indicating that the two APC5 components act largely redundantly at this stage.

Therefore, in contrast to the situation in Drosophila, where some APC/C substrates are turned over in embryos harboring a genetic null mutation in the sole APC5 gene ida (Bentley et al. 2002), the available evidence indicates that in C. elegans APC5 is essential for canonical APC/C function.

Mutual antagonism between the anaphase promoting complex and the spindle assembly checkpoint:

In most systems, engagement of the SAC results in a drastic delay of mitotic progression. By contrast, in C. elegans embryos, SAC engagement causes only a twofold prolongation in M phase progression (Encalada et al. 2005; Essex et al. 2009), similar to the minor delay observed in other early embryonic systems (Minshull et al. 1994). Moreover, partial inactivation of the APC/C also results in merely a marginal delay during mitosis in C. elegans embryos (Tarailo et al. 2007). Importantly, we found that engaging the SAC in embryos with compromised APC/C function results in a substantial prolongation of M phase, which lasts approximately seven times longer than in the wild type. At least two models could explain the mechanism leading to prolonged M phase duration in APC/C mutants. First, the SAC, which negatively regulates the APC/C, may further compromise the already crippled APC/C. Since APC/C governs mitotic exit, the combination of SAC engagement and compromised APC/C may provoke the extended duration of mitosis. In this first model, one might expect that the overall delay should correspond to the additive effect of these two factors. However, whereas the mitotic delay compared to the wild type is twofold upon SAC engagement and 1.3-fold in such-1(t1668) embryos with a bipolar spindle, it is sevenfold in such-1(t1668) mutant embryos with an engaged checkpoint. Because of this synergistic effect, we favor a second model in which the APC/C negatively regulates the SAC in C. elegans embryos (Figure 7A), as has been proposed from biochemical and cell biological experiments in human cells (Reddy et al. 2007). During SAC engagement in the wild type, the ubiquitination by the APC/C of substrates such as securin and cyclin B is inhibited, due to sequestration of Cdc20 by a MAD2- and/or BubR1-containing complex. At the same time, Cdc20 is turned over, and this continuous degradation is dependent on the SAC as well as on the APC/C (Pan and Chen 2004; Reddy et al. 2007; Nilsson et al. 2008). Once the SAC is inactivated, Cdc20 becomes available to fully activate the APC/C, thus enabling efficient ubiquitination of mitotic substrates. Both Cdc20 accumulation and Cdc20 liberation from the MAD2- and/or BubRI-containing complex have been proposed to be instrumental for full APC/C activation at this stage (Pan and Chen 2004; Nilsson et al. 2008). Our observations support the notion that the APC/C participates in inactivating the SAC (Reddy et al. 2007), and thus that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos (Figure 7A). When APC/C is compromised, this negative feedback mechanism is dampened, leading to prolonged SAC engagement and extended mitosis (Figure 7B).

Figure 7.—

Working model highlighting the mutual antagonism between the SAC and APC/C^Cdc20. (A) In the wild type, SAC engagement inhibits APC/C^Cdc20 activity; APC/C^Cdc20 reciprocally negatively regulates the SAC. Increased APC/C^Cdc20 activity is required to turn off the SAC and allow sister chromatid segregation and M phase exit. (B) In conditions where APC/C^Cdc20 activity is low, the negative regulation from the APC/C^Cdc20 is weakened, which results in a substantially prolonged engagement of the SAC, and hence increased M phase duration.

Mechanisms of SAC inactivation by the APC/C:

How does the APC/C participate in inactivating the SAC in C. elegans embryos? We found that reduction of ubiquitination by partial depletion of the E1 enzyme UBA-1 also provokes excess SAC engagement upon monopolar spindle assembly. The relevant ubiquitination event may influence the function or the half-life of the substrate. Regardless, our observations are in line with those in human cells, where ubiquitination of substrates promotes mitotic exit after SAC engagement (Reddy et al. 2007; Summers et al. 2008; Garnett et al. 2009; Williamson et al. 2009). In this context, the APC/C together with the E2 UbcH10 monoubiquitinates substrates, which are subsequently elongated by the E2 Ube2S, leading to substrate degradation (Summers et al. 2008; Garnett et al. 2009; Williamson et al. 2009; Wu et al. 2010). Interestingly, Ube2S depletion does not affect cell cycle progression or cyclin B degradation in unperturbed cells, but becomes essential for SAC inactivation when microtubules are disrupted (Garnett et al. 2009). This raises the possibility that Ube2S is a specific SAC silencing E2 enzyme in humans. A Ube2S homolog has been identified but not yet studied in Drosophila, but sequence analysis did not reveal a C. elegans counterpart (Jones et al. 2002), raising the possibility that other mechanisms are at play in this respect in nematodes.

Which critical substrates might be ubiquitinated by the APC/C to inactivate the SAC in C. elegans? A plausible candidate is Cdc20 itself. Cdc20 is ubiquitinated by the APC/C and thereby targeted for degradation during SAC engagement in human cells (Pan and Chen 2004; Reddy et al. 2007; Nilsson et al. 2008). Moreover, Cdc20 ubiquitination reduces binding to MAD2 (Reddy et al. 2007), and this has been proposed to constitute a mechanism whereby the checkpoint is inactivated. However, the fact that a nonubiquitylatable form of Cdc20 drives checkpoint-arrested human cells out of mitosis challenged these views (Nilsson et al. 2008), such that the exact contribution of Cdc20 ubquitination remains to be resolved.

Our findings underscore the essential role of FZY-1/Cdc20 during prolonged SAC activation in C. elegans embryos. The fzy-1(av15) allele used to suppress the drastic M phase delay was proposed to affect a MAD2 binding domain (Stein et al. 2007), which likely is critical for communicating the status of the SAC to the APC/C. Because Cdc20 levels are cell-cycle regulated and its overexpression overrides the SAC in yeast, it appears that Cdc20 levels or activity control the strength of SAC engagement (Prinz et al. 1998; Schott and Hoyt 1998; Pan and Chen 2004). In early C. elegans embryos, FZY-1/Cdc20 levels might be more stable, as both interphase and M phase cells stain brightly for FZY-1 (A. Bezler and P. Gönczy, unpublished observation; Kitagawa and Rose 1999). In these rapidly dividing cells, degradation of FZY-1/Cdc20 at the end of the cell cycle might not occur; instead, there might be a slow decline, as observed for cyclin B in Drosophila syncytial embryos (Edgar et al. 1994).

In conclusion, our findings lead us to propose that the APC/C negatively regulates the SAC in early C. elegans embryos, suggesting that mutual antagonism between the APC/C and the SAC modulates the duration of mitosis during the cleavage divisions of a developing organism.