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.

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.

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.

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).

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).

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.

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.