Tunquist BJ , Eyers PA , Chen LG , Lewellyn AL , Maller JL .

DK28353-21 NIDDK NIH HHS , R01 DK028353 NIDDK NIH HHS

	Figure 1. Analysis of recombinant Mad1 and Mad2. (A) Aliquots of Mad1, Mad2, and Mad2 R133A recombinant proteins used in experiments were analyzed by SDS-PAGE and visualized by Coomassie staining. 2-, 1-, 0.5-, and 0.1-μg amounts of BSA are also visualized on the gel as standards for determination of protein concentration. The entire gel is shown. (B–D) 1/50 of the volume of Mad1 or Mad2 R133A used in A, and 1/100 of the volume of Mad2 were loaded onto an SDS-PAGE gel for Western blotting. The entire gel is shown. (B) Samples immunoblotted with anti-Mad1 antibodies. GST-Mad1 and Mad1 degradation products are shown. (C) Samples immunoblotted with anti-GST antibodies (Santa Cruz Biotechnology, Inc.). GST-Mad1 protein and NH2-terminal degradation products of Mad1 containing the GST tag are indicated. In addition, GST protein is indicated. (D) Samples immunoblotted with anti-Mad2 antibodies. Recombinant Mad2 and Mad2 R133A proteins are indicated. (E) Cleavage arrest in blastomeres caused by Mad1 or Mad2. One blastomere of a two-cell embryo was injected with 50 ng BSA (control), Mad1, Mad2, or Mos protein. Development was monitored closely with a dissecting microscope and the embryos were photographed when control embryos had reached stage 7. Metaphase spindles were visualized by confocal microscopy from embryos in which both blastomeres at the two-cell stage were injected with Mad1, Mad2, or Mos. Red indicates α-tubulin, green indicates DNA, and yellow indicates an overlapping signal. (F) Oligomerization of Mad2 is required for CSF arrest. Blastomere injection assays were performed as in A, except that one blastomere was also injected with Mad2 R133A. The embryos were photographed when control embryos had reached stage 6.
	Figure 2. Mad2 is required for establishment of CSF arrest by Mos. (A–E) Histone H1 kinase activity. Extracts in interphase at 4°C were treated with protein A Dynabeads with or without bound anti-Mad2 antibodies to immunodeplete endogenous Mad2 protein (G), and then were supplemented with 500 sperm nuclei/μl. (A) Mock-depleted extract supplemented with Mos injection buffer. (B) Mock-depleted extract supplemented with Mos protein (60 ng/μl). (C) Mad2-depleted extract supplemented with Mos protein. (D) Mad2-depleted extract supplemented with Mos and recombinant Mad2 protein (30 ng/μl). (E) Mad2-depleted extracts supplemented with Mos and recombinant Mad2 R133A protein (30 ng/μl). Cycling was initiated by warming to 22°C, and aliquots of the extracts were monitored for histone H1 kinase activity. The “M” bar in each panel refers to the length of M phase as determined by DAPI staining (F). (F) Effect of Mad2 depletion on nuclear morphology in cycling extracts. The samples shown in A–E were monitored for progression of the cell cycle by DAPI staining and fluorescence microscopy at the indicated times. Nuclei in all samples entered mitosis by 15 min. Addition of Mos at 0 min caused a prolonged M phase in the presence, but not the absence, of Mad2; arrest by Mos could be restored by the addition of wild-type, but not R133A Mad2 protein. (G) Immunodepletion of Mad2. Affinity-purified rabbit pAb to Mad2 was bound to protein A Dynabeads and incubated with a cycling extract in interphase for 45 min at 4°C (lanes 3–5). Mock-depleted extract (lane 1 and lane 2) was generated by incubation of extract with beads bound with control IgG. The supernatants were collected and immunoblotted for Mad2.
	Figure 3. Mad1 is required for establishment of CSF arrest by Mos. (A–D) Histone H1 kinase activity. Extracts in interphase at 4°C were treated with Dynabeads coupled to protein A with or without bound anti-Mad1 antibodies to immunodeplete endogenous Mad1 (F), then supplemented with 500 sperm nuclei/μl extract. (A) Mock-depleted extract supplemented with Mos injection buffer. (B) Mock-depleted extract supplemented with 60 ng/μl Mos protein. (C) Mad1-depleted extract supplemented with Mos protein. (D) Mad1-depleted extract supplemented with 60 ng/μl Mos and 10 ng/μl Mad1 protein. Cycling was initiated by warming to 22°C, and aliquots of the extracts were monitored for histone H1 kinase activity. The “M” bar in each panel refers to the length of M phase as determined by DAPI staining (E). (E) Effect of Mad1 depletion on nuclear morphology in cycling egg extracts. The samples shown in A–D were monitored for progression of the cell cycle by DAPI staining and fluorescence microscopy at the indicated times. Nuclei in all samples entered mitosis by 30 min, and extracts treated with control beads exited from M phase by 45 min. Addition of Mos at 0 time (45 min after CSF release) caused a prolonged M phase in the presence, but not the absence, of Mad1; arrest by Mos could be restored by the addition of recombinant Mad1 protein. (F) Immunodepletion of Mad1. Rabbit antibodies to Mad1 were bound to protein A Dynabeads and incubated with a cycling extract in interphase for 45 min at 4°C (lane 3 and lane 4). Mock-depleted extracts (lanes 1 and 2) were generated by incubation of extract with beads bound with control IgG. The supernatants were collected and immunoblotted for Mad1.
	Figure 4. Effect of Mad1 and Mad2 addition to cycling egg extracts. Mad1 or Mad2 was added to a cycling extract at a final concentration of 10 ng/μl extract (Mad1) or 30 ng/μl extract (Mad2), and cell cycle progression was monitored by DAPI staining. Cell cycle progression was not altered by Mad1 or Mad2 relative to the control, whereas addition of 60 ng/μl Mos did cause M phase arrest. The total concentration of Mad1 and Mad2 in the extract was identical to that in the reconstitution experiments in Fig. 2 and Fig. 3.
	Figure 5. Requirement of Mad1 and Mad2 for CSF arrest in vivo. (A and B) Stage VI oocytes were microinjected with ∼20 ng of affinity-purified antibody against Mad2 protein, or affinity-purified antibody depleted of Mad2 immunoreactivity (flow-through IgG). Oocytes were treated with progesterone, and at the indicated times after germinal vesicle breakdown, oocytes were homogenized and clarified extracts were assayed for histone H1 kinase activity (A) or immunoblotted for the level of cyclin B2 (B) as described in the Materials and methods section. (C and D) Oocytes microinjected with Mad1 immune serum or Mad1 preimmune serum (control) were collected, and assayed for histone H1 kinase activity (C) or the level of cyclin B2 (D) at the indicated times.
	Figure 6. Analysis of Mad1 and Mad2 for maintenance of CSF arrest. (A) Overexpression of Mad1 or Mad2 blocks release from CSF arrest by calcium. 30-μl CSF extracts were supplemented with 6 μg BSA (control), 6 μg Mad1, or 6 μg Mad2 protein and incubated for 20 min at 22°C before addition of 500 μM calcium or 1 μM microcystin-LR. Aliquots were taken 1, 10, or 30 min after calcium or microcystin addition, and histone H1 kinase activity was measured. The panel shows an autoradiograph of radiolabeled histone H1 protein. (B) Mad1, but not Mad2, is required for maintenance of CSF arrest. The histone H1 kinase activity (autoradiograph; top), and cyclin B2 levels (immunoblot; bottom) of a CSF-arrested extract prepared from unfertilized eggs without added nuclei was monitored for 45 min after mock, Mad1, Mad2, or Mad1 plus Mad2 immunodepletion before addition of calcium sufficient to cause CSF release (500 μM). (C) Immunoblot of supernatants (extract) or beads after the immunodepletion of Mad1, Mad2, or both proteins shown in B. Aliquots of the indicated immunodepleted samples at the 15-min time point (B) were immunoblotted for Mad1 or Mad2 (top two panels) in the depleted supernatant or on the beads. Also shown are immunoblot of supernatants or beads for Cdc20 after immunodepletion of Mad1, Mad2, or both proteins (bottom two panels). To confirm that the designated immunostaining band was indeed Cdc20, anti-Cdc20 antibody was pre-blocked with recombinant Cdc20 protein (bottom) and used for immunoblotting. The increased electrophoretic mobility shift of Cdc20 upon exit from M phase has been previously shown to reflect altered phosphorylation (Taieb et al., 2001). (D) Mad1-depleted extract supplemented with GST-Mad1 protein. The histone H1 kinase activity (autoradiograph; top), and cyclin B2 levels (immunoblot; bottom) of a CSF-arrested extract prepared as in (B) was monitored for 45 min after mock depletion, Mad1 depletion, or Mad1 depletion supplemented with GST-Mad1 protein (50 ng/μl extract). This level of GST-Mad1 had no effect on the ability of calcium to cause CSF release when added 45 min after warming.

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