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First published online 20 November 2002
doi: 10.1242/jcs.00232

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Regulation of meiotic progression by the meiosis-specific checkpoint kinase Mek1 in fission yeast

Livia Pérez-Hidalgo, Sergio Moreno and Pedro A. San-Segundo*

Centro de Investigación del Cáncer, CSIC/University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain



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  		Fig. 1. Fission yeast Mek1 belongs to the family of Cds1/Rad53/Chk2 checkpoint kinases and is expressed only in meiotic cells. (A) Alignment of the protein sequences of S. cerevisiae Mek1 (ScMek1), S. pombe Mek1 (SpMek1) and S. pombe Cds1 (SpCds1). Identities are highlighted in black and conservative substitutions in gray. Numbers at the left represent amino-acid positions. Alignment and shading were performed with ClustalW 1.8 and Boxshade 3.21, respectively, at the BCM Search Launcher (http://searchlauncher.bcm.tmc.edu/). (B) Phylogenetic tree of the family of FHA checkpoint kinases. The clustal method with PAM250 residue table was used. Sp, S. pombe; Sc, S. cerevisiae; Mm, M. musculus; Hs, H. sapiens; Xl, X. laevis; Ce, C. elegans. A schematic representation of the functional motifs in this protein family (with the exception of Rad53, which contains two FHA domains) is shown. (C) PCR amplification of S. pombe mek1+ using primers mek1-N and mek1-C (small arrows; see Materials and Methods). The following DNA templates were used: cDNA from vegetative S. pombe cells (Veg), cDNA from meiotic cells (S964) at 2 hours and 3 hours in meiosis (Mei 2h and Mei 3h, respectively) and S. pombe genomic DNA.

 


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  		Fig. 2. Mek1 is a meiosis-specific nuclear protein. (A) Schematic representation of pat1-driven synchronous meiosis. Vegetatively growing cells are blocked in G1 by nitrogen starvation during ~14 hours and then induced to enter meiosis synchronously by inactivating the Pat1 kinase at 34°C (time 0 in B, C and D). The approximate timing of the major meiotic landmarks, as determined in (B), is indicated. (B) Synchronous meiosis of strain S964. Left panel, DNA content measured by FACS analysis. The period in which premeiotic DNA replication takes place is indicated (meiS). Note that the 1C peak that appears after 7 hours corresponds to free spores that are released from asci owing to sonication during the preparation of cells for FACS. Right panel, meiotic progression was followed by DAPI staining of nuclei and sporulation by microscopic observation of asci. The peaks of meiosis I (MI), meiosis II (MII) and spore formation (Spo) are indicated. (C) Northern blot analysis of mek1+ expression during the synchronous meiosis of strain S964 shown in B. 18S rRNA levels are shown as a loading control. (D) Western blot analysis of Mek1-HA production during a synchronous meiosis in strain S1294. Tubulin is presented as a loading control. (E) Immunofluorescence analysis of cells from strain S1294 (mek1-HA), after 3 hours of induction of meiosis, stained with DAPI (blue) and anti-HA antibodies (red). The merged image is presented in the right column. Three representative cells are shown.

 


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  		Fig. 3. The mek1 mutant is defective in spore viability and meiotic recombination. (A) Distribution of tetrad types. The percentages of tetrads with 4, 3, 2, 1 and 0 viable spores (4-sv, 3-sv, 2-sv, 1-sv and 0-sv, respectively) are represented. Tetrads were dissected from crosses between wild-type (S778xS781) and mek1 strains (S1291xS1292) after 2 days on MEA plates. (B) The frequency of intergenic meiotic recombination was measured on the leu1-his5 interval on chromosome II by random spore analysis of crosses between wildtype (PN22 x S1285) and mek1 (S1300xS1301) strains.

 


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  		Fig. 4. Mek1 regulates entry into meiosis I. (A) The mek1 mutant enters meiosis I faster than wildtype. Kinetics of meiotic progression of wildtype (S964; upper panel) and mek1 (S1293; lower panel) was followed by DAPI staining of nuclei. Note that the peak of meiosis I (MI) occurs ~30 minutes earlier in the mek1 mutant. A representative time course is presented, but the experiment was repeated at least three times, the same results being obtained. (B) FACS analysis of the meiotic time courses shown in A. Note that the timing of premeiotic S phase (meiS) is the same in both the wildtype (upper panel) and mek1 mutant (lower panel). (C,D,E) High levels of Mek1 delay entry into meiosis I. (C) Schematic representation of the experimental procedure used for overexpression of mek1+ from the thiamine-repressible nmt1 promoter during meiosis in strain S1298. T, thiamine; N, nitrogen. (D) FACS analysis of meiotic time courses of S1298 in the presence (upper panel) or in the absence (lower panel) of thiamine. (E) Kinetics of meiotic progression monitored by DAPI nuclear staining during meiotic time courses of strain S1298 in the presence of thiamine (nmt-mek1+ OFF) or in the absence of thiamine (nmt-mek1+ ON). Note that the peak of meiosis I (MI) takes place ~1 hour later when Mek1 is overproduced.

 


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  		Fig. 5. Ectopic overexpression of mek1+ in vegetative cells causes Cdc25-dependent G2/M cell cycle arrest. (A) Wild-type cells (PN22) transformed with the pREP3X vector or with plasmid pSS123 (nmt-mek1+) were streaked out on plates containing thiamine (nmt1 promoter OFF) or lacking thiamine (nmt1 promoter ON) and incubated for 3 days at 30°C. (B) Cells from strain S1297, which contain an integrated nmt1-mek1-GFP construct, were incubated in nmt1-repressing conditions (OFF) or nmt1-inducing conditions (ON) for 24 hours. Nuclei were stained with DAPI. Backlight reveals cell bodies. (C) Wild-type (PN22), wee1-50 (S145), cdc2-3w (S176) and cdc2-3w cdc25{Delta} (S898) cells transformed with pSS124 were grown on plates containing or lacking thiamine (nmt-mek1+ OFF and nmt-mek1+ ON, respectively). For wildtype, cdc2-3w and cdc2-3w cdc25{Delta}, plates were incubated at 30°C, whereas wee1-50 cells were incubated at 36°C to inactivate Wee1 function. Microcolonies were photographed after ~30 hours. Note that cdc25{Delta} cells do not elongate in response to mek1+ overexpression. (D) Strains S1297 (wildtype) and S1302 (cdc25-9A), which contain nmt1-mek1-GFP integrated in the genome were incubated in the absence (OFF) or in the presence (ON) of thiamine for 22 hours. Cells were visualized at the fluorescence microscope using a GFP filter. Backlight reveals cell bodies. Note that in the absence of thiamine Mek1-GFP accumulates in the nucleus of both wildtype and cdc25-9A, but cdc25-9A cells elongate considerably less than wildtype; in addition, cdc25-9A binucleate dividing cells are observed frequently.

 


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  		Fig. 6. The Mek1-dependent meiotic recombination checkpoint regulates phosphorylation of Cdc2 on Tyr15. (A) Meiotic time courses of wild-type (S964), meu13 (S1295) and meu13 mek1 (S1296) strains. The percentage of cells that have undergone the first meiotic division (2 nuclei) or both meiotic divisions (3/4 nuclei) is shown. Note that the meu13 mutant shows a delay (~30 minutes) in entering meiosis I that is alleviated by the mek1 mutation. Arrows pointing to the percentage of binucleate cells at the 4 hour time point are shown to highlight the meu13 delay. (B) Western blot analysis of the meiotic time courses shown in A, also including a mek1 strain (S1293; see Fig. 4A), using anti-phopho-Cdc2(Tyr15) and anti-Cdc2 antibodies. Note that phosphorylation of Cdc2 on Tyr15 persists longer when the meiotic recombination checkpoint is triggered (i.e., in the meu13 mutant) but not when the checkpoint is inactivated by mutation of mek1+.

 


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  		Fig. 7. The meiotic recombination checkpoint is impaired in the cdc25-9A phosphorylation site mutant. Meiotic time courses of cdc25-9A (S1308; top panel), meu13 (S1306; middle panel) and meu13 cdc25-9A (S1307, lower panel) strains. The percentages of cells that have undergone the first meiotic division (2 nuclei) or both meiotic divisions (3/4 nuclei) are shown. Arrows pointing to the percentage of binucleate cells at the 4 hour time point are presented to highlight the suppression of the meu13 delay by the cdc25-9A mutation.

 


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  		Fig. 8. A model for the meiotic recombination checkpoint pathway in fission yeast. See text for details.

 

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© The Company of Biologists Ltd 2003