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Fission yeast mfr1 activates APC and coordinates meiotic nuclear division with sporulation

Miguel A. Blanco*, Laetitia Pelloquin* and Sergio Moreno{ddagger}

Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno, 37007 Salamanca, Spain
* These two authors contributed equally to this work



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  		Fig. 1. Fission yeast mfr1 is related to ste9/srw1. (A) mfr1 belongs to the Fizzy-related family of APC activators. Phylogenetic tree using the clustal method with PAM250 residue weight table. (B) Protein sequence comparison between ste9 and mfr1. Black boxes indicate identity; asterisks indicate related amino acids. The homology between ste9 and mfr1 extends beyond the seven WD repeats, including domains I and II previously described (Cebolla et al., 1999).

 


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  		Fig. 2. mfr1+ is expressed exclusively during meiosis. Cells of the diploid strain Sp966 were presynchronized in G1 by nitrogen starvation for 14 hours at 25°C. Nitrogen was added and the culture was incubated at 34°C to inactivate the pat1-114 temperature-sensitive protein kinase. (A) Northern and western blots showing mfr1+ mRNA and protein levels. Cdc13 and cdc2 protein levels are shown. Exp, mitotic exponentially growing cells before nitrogen starvation. (B) FACS analysis. Pre-meiotic S-phase occurred between 2 and 3 hours. Cells with 1C DNA content observed after 6.5 hours correspond to haploid spores that are released when cells are sonicated before flow cytometry. (C) Percentage of cells with 1, 2 and 4 nuclei and percentage of asci. Meiosis I occurred between 4 and 5 hours, meiosis II between 5 and 6 hours and sporulation after 7.5 hours.

 


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  		Fig. 3. mfr1+ is required for sporulation. A wild-type diploid (Sp785) and a mfr1{Delta}/mfr1{Delta} mutant (Sp963) were sporulated on agar plates containing malt extract (sporulation medium). (A) Photographs of cells after 48 hours using interference microscopy (top) and DAPI staining (bottom). In the mfr1{Delta}/mfr1{Delta} mutant most cells contained four nuclei, as in the wild type. These nuclei were difficult to photograph because they were on different focal planes. (B) Percentage of asci after 48 hours with 4, 3, 2, 1 and 0 spores.

 


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  		Fig. 4. The mfr1{Delta}/mfr1{Delta} mutant completes meiosis II but fails to undergo sporulation. The strains h-/h- pat1-114/pat1-114 (Sp964) and h-/h- pat1-114/pat1-114 mfr1{Delta}/mfr1{Delta} (Sp965) were synchronized through meiosis as indicated in Fig. 2. (A) FACS analysis. Pre-meiotic S-phase took place between 2 and 3 hours in both cases. Haploid spores (as cells with 1C DNA content) were observed in the control cells after 7 hours but not in the mfr1{Delta}/mfr1{Delta} mutant. Exp, exponentially growing cells before nitrogen starvation. (B) Percentage of cells with 1, 2 and 4 nuclei and of asci with spores during the experiment. Spores were not observed in the mfr1{Delta}/mfr1{Delta} strain during this experiment. (C) Spindle-pole body (SPB) staining with anti-sad1 antibodies and DAPI staining in cells undergoing meiosis I (M I) and meiosis II (M II). The two cells shown in the right panels in each case have modified SPBs.

 


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  		Fig. 5. mfr1 colocalizes and interacts with APC during meiosis. (A) Cells of the diploid strain Sp967, containing mfr1 tagged with three copies of the HA epitope and lid1(APC4) tagged with 9 copies of the myc epitope, were induced to undergo synchronous meiosis as indicated in Fig. 2. After 6 hours, cells were fixed and stained with anti-HA monoclonal antibodies, anti-myc polyclonal antibodies, and with DAPI. Nuclei 1 and 2 are in meiosis I. Cells in the right panels are control cells (Sp964) lacking the HA and the myc tags stained with anti-HA and anti-myc antibodies. (B) The diploid strains Sp967 and the control Sp966 with identical genotype except that it is wild type for lid1+ were synchronized in meiosis. Samples were taken at 5.5, 6, 6.5 and 7 hours for immunoprecipitation with anti-HA or anti-myc antibodies. Immunoprecipitates were run on SDS-PAGE gels and probed with anti-HA or anti-myc antibodies. Western blots of total extracts were probed with anti-myc and anti-HA antibodies to check the levels of the tagged proteins. Western blot with anti-cdc2 is shown as loading control. * IgG heavy chain.

 


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  		Fig. 6. Cdc13 cyclin is stabilized in the mfr1{Delta}/mfr1{Delta} mutant. (A) Strains h-/h- pat1-114/pat1-114 (Sp964) and h-/h-pat1-114/pat1-114 mfr1{Delta}/mfr1{Delta} (Sp965) were synchronized through meiosis as indicated in Fig. 2. Samples were taken every 30 minutes and protein extracts were analyzed by western blot with antibodies to cig1, cdc13 and cdc2. Exp, exponentially growing cells before nitrogen starvation. (B) Cdc2 protein kinase assays after immunoprecipitation with cdc2 antibodies. Samples were taken every hour.

 


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  		Fig. 7. Expression of non-degradable cdc13-des2 destruction box mutant inhibits sporulation. The diploid strain h-/h- pat1-114/pat1-114 (Sp964) was transformed with pJK148-cdc13+ or pJK148-cdc13-des2 to generate the strains Sp969 and Sp970, respectively. Single-copy integrants at the leu1 locus were isolated and induced to undergo a synchronous meiosis. (A) Photographs of cells after 9 hours at 34°C. (B) Percentage of asci with 4, 3, 2, 1 and 0 spores.

 

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