report
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Cell Cycle 10:6, 951-955; March 15, 2011; © 2011 Landes Bioscience
Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I Andrej Dudas, Shazia Ahmad and Juraj Gregan* Department of Chromosome Biology; Max F. Perutz Laboratories; University of Vienna; Vienna, Austria
Key words: meiosis, chromosome segregation, recombination, kinetochore, Sgo1, fission yeast
The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I chromosomes which ensure the reductional nature of chromosome segregation. Here we show that sister chromatids frequently segregate to opposite poles during meiosis I in fission yeast cells that lack both chiasmata and the protector of centromeric cohesion Sgo1. Our data are consistent with the notion that sister kinetochores are frequently bi-oriented in the absence of chiasmata and that Sgo1 prevents equational segregation of sister chromatids during achiasmate meiosis I.
Introduction
Results
During meiosis, two successive rounds of chromosome segregation (meiosis I and meiosis II), which follow a single round of DNA replication generate haploid gametes from diploid precursor cells (reviewed in refs. 1–4). Three major features of meiosis I chromosomes ensure the reductional nature of chromosome segregation during meiosis I. The first is the formation of chiasmata, which provide the physical link between homologous chromosomes. Chiasma formation depends on meiotic recombination, which is initiated by Spo11/Rec12-induced double-strand breaks (DSBs) (reviewed in refs. 5–9). The second meiosis I-specific feature is the attachment of sister kinetochores to microtubules emanating from the same spindle pole (mono-orientation) (reviewed in refs. 10–12). Finally, cohesion between sister centromeres must be protected during meiosis I. The protection of centromeric cohesion is mediated by the conserved Sgo1/MEI-S332 proteins that recruit a specific form of protein phosphatase 2A (called PP2A-π) to centromeres.13-21 The Sgo1/PP2A complex then protects centromeric cohesin from separase cleavage by opposing phosphorylation of Rec8, the meiosis-specific alphakleisin subunit of cohesin.22-27 Coordination of these processes is crucial for proper segregation of chromosomes during meiosis and defects in these processes may lead to meiotic aneuploidy, which is the leading cause of miscarriages and genetic disorders such as Down syndrome in humans.28-30
In order to identify mutants defective in the mono-orientation of sister kinetochores during meiosis I, Yokobayashi and Watanabe performed an elegant genetic screen in a haploid S. pombe strain that undergoes a single meiotic division and produces two spored-asci immediately after meiosis I.31 These spores are mostly non-viable, because sister chromatids co-segregate to the same pole (reductional-like segregation) and only occasionally all three chromosomes segregate to the same nucleus.32 Yokobayashi and Watanabe reasoned that mutations affecting mono-orientation of sister kinetochores should shift chromosome segregation from reductional-like to equational and produce viable spores. This screen led to the identification of Moa1 as well as other proteins involved in sister-chromatid cohesion.31 However, this screen was not ideal for identification of factors required specifically for the mono-orientation process, because centromeric cohesion which remains intact during the first meiotic division, may prevent segregation of bi-oriented sister chromatids to the opposite poles in mutants defective in the mono-orientation, but not in the protection of centromeric cohesion. We therefore decided to carry out a new screen in a haploid strain carrying an additional mutation (sgo1Δ), which renders the centromeric cohesion sensitive to separase cleavage during meiosis I (reviewed in refs. 17 and 33). In the absence of Sgo1, centromeric cohesion is resolved at the onset of anaphase I and this should allow segregation of bi-oriented sister chromatids to the opposite poles. However, we found that this strain frequently segregated sister chromatids to opposite poles during meiosis I and produced spores with high viability (Fig. 1A). This is surprising, because during diploid meiosis,
*Correspondence to: Juraj Gregan; Email:
[email protected] Submitted: 11/18/10; Revised: 02/02/11; Accepted: 02/07/11 DOI: 10.4161/cc.10.6.15032 www.landesbioscience.com
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Figure 1. Sgo1 is required for co-segregation of sister chromatids during achiasmate meiosis I. (A) Analysis of sister-chromatid segregation during haploid meiosis I. The indicated strains were sporulated, stained with Hoechst 33342 and examined under the fluorescence microscope. Segregation of chromosome I (labeled with lys1-GFP) was scored in 100 dyads. Spore viability was determined by dissection of dyads (80 spores were analyzed for each strain). The genotypes of strains are listed in the Table 1. (B) Analysis of sister-chromatid segregation during anaphase I. The indicated strains were sporulated, fixed, stained with Hoechst 33342 and antibodies against tubulin and GFP, and examined under the fluorescence microscope. Segregation of chromosome I (heterozygous lys1-GFP) and chromosome II (heterozygous cen2-GFP) was scored in 100 anaphase I cells. In wild-type cells, we attribute the rare cases of segregation to opposite poles of sister lys1-GFP sequences to recombination taking place between cen1 and lys1. (C) Analysis of sister chromatid cohesion during meiotic prophase. Cells were prepared as in (B) and sister chromatid cohesion was assayed in horsetail nuclei using strains where one copy of either chromosome I (heterozygous lys1-GFP) or chromosome II (heterozygous cen2-GFP) was labeled with GFP.
sister chromatids efficiently co-segregate to the same pole in the absence of Sgo1.17,18 We thus conclude that Sgo1 is required for efficient co-segregation of sister chromatids during haploid meiosis I. The observed equational segregation of sister chromatids in haploid sgo1Δ cells might be due to a defect in the protection of centromeric cohesion, because we observed a similar phenotype in cells lacking Par1 subunit of the PP2A, which is also required for the protection of centromeric cohesion during meiosis I (Fig. 1A).14,34 Although this unexpected finding prevented us from pursuing our genetic screen, it raised an interesting question: why
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is Sgo1 required for co-segregation of sister chromatids during haploid meiosis I, but not during diploid meiosis I? One obvious difference between haploid and diploid meiosis is that homologous chromosomes are linked by chiasmata in diploid meiosis. We therefore decided to test the possibility that Sgo1 is required for co-segregation of sister chromatids during meiosis I in the absence of chiasmata. We scored segregation of sister chromatids during diploid meiosis in sgo1Δ mutant strain lacking chiasmata due to rec12Δ mutation.35 Indeed, sister chromatids frequently segregated to opposite poles in sgo1Δ rec12Δ double mutant
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(Fig. 1B). Similarly, we observed increased equational segregation of sister chromatids in rec12Δ mutant cells lacking Ppa2 subunit of the PP2A, which is also required for the protection of centromeric cohesion during meiosis I (Fig. 1B).14,34 This equational segregation is likely caused by the absence of chiasmata and not due to a potential role of Rec12 at centromeres, because we observed a similar phenotype in mde2Δ mutant cells, which are also defective in chiasma formation13 (Fig. 1B). The observed equational segregation of sister chromatids in sgo1Δ rec12Δ and ppa2Δ rec12Δ mutant cells is unlikely due to loss of sister chromatid cohesion prior to the onset of anaphase I, because sister chromatids (visualized by heterozygous cen2-GFP or lys1-GFP) were cohesed during meiotic prophase (horsetail stage) (Fig. 1C) and metaphase I (data not shown). We therefore conclude that, in fission yeast, Sgo1 is required for efficient co-segregation of sister chromatids during achiasmate meiosis I. Discussion We envision the following two models, which are not mutually exclusive, of how Sgo1 ensures co-segregation of sister chromatids in the absence of chiasmata. First, Sgo1 might be directly involved in the mono-orientation of sister kinetochores during achiasmate meiosis I. However, this possibility is less likely, because neither Sgo1 nor PP2A is required for the mono-orientation in diploid meiosis when chiasmata are present.17,18 The second possibility is that sister kinetochores are frequently bi-oriented in achiasmate meiosis I. However, bi-oriented sister chromatids are not able to segregate to opposite poles, because centromeric cohesion resists pulling forces of the spindle. In the absence of Sgo1, centromeric cohesion is resolved at the onset of anaphase I and this allows segregation of bi-oriented sister chromatids to opposite poles. Consistent with this model is our analysis of lagging chromosomes on anaphase I spindles in rec12Δ mutant cells. We analyzed those anaphase I cells where chromosome II was lagging and observed that sister centromeres (labeled by heterozygous cen2-GFP32) were separated in 15 out of 50 analyzed cells. In all 15 cells sister centromeres were separated along the spindle axis, suggesting that these chromosomes were lagging due to bi-orientation of sister kinetochores (Fig. 2). Several lines of evidence support the role of chiasmata in orientation of sister kinetochores during meiosis I. Previous studies in fission yeast showed that although recombination between homologous chromosomes is not absolutely required for the co-segregation of sister chromatids during meiosis I, it increases the fidelity of this process.32 Moreover, bi-orientation of achiasmate chromosomes has been observed during anaphase I in mammalian and plant cells.36-41 However, sister kinetochores are mono-oriented during meiosis I in S. cerevisiae mutant cells defective in chiasma formation and protection of centromeric cohesion (spo11Δ rts1Δ).15 Thus, it is possible that bi-orientation of achiasmate chromosomes during meiosis I is a general phenomenon, however, some organisms posses mechanisms that ensure mono-orientation of sister kinetochores during meiosis I in the absence of chiasmata. Although our data are consistent with the notion that Sgo1-mediated protection of centromeric cohesion prevents segregation of
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Figure 2. Lagging sister centromeres are frequently separated along the spindle axis in anaphase I cells lacking chiasmata. The indicated strains were sporulated, fixed, stained with Hoechst 33342 and antibodies against tubulin and GFP, and examined under the fluorescence microscope. Anaphase I cells with lagging cen2-GFP signals (representing chromosome II) were scored (n = 50).
bi-oriented sister chromatids to opposite poles, other studies indicate that protection of centromeric cohesion does not operate when sister kinetochores are bi-oriented.42-44 On the other hand, ectopic co-expression of Rec8 and Sgo1 in vegetative cells is sufficient to prevent anaphase, suggesting that under these conditions bi-orientation does not prevent Sgo1 from protecting Rec8 from separase cleavage.17 Therefore, further analysis is required to decipher how Sgo1 ensures co-segregation of sister chromatids during achiasmate meiosis I. An important question which remains to be answered is how chiasmata affect orientation of sister kinetochores. One possibility is that processes leading to chiasma formation alter kinetochore architecture such that sister kinetochores are preferentially attached to microtubules emanating from the same spindle pole. An alternative, but not mutually exclusive, explanation is that the physical linkage between homologous chromosomes mediated by chiasmata promotes mono-orientation of sister kinetochores. Elucidating the molecular mechanisms of how chiasmata affect orientation of sister kinetochores during meiosis I will be an important aim of future studies. Materials and Methods The immunofluorescence, microscopy, media and growth conditions were as previously described in references 33 and 46. Images were processed using Photoshop software (Adobe Systems). Genes were deleted according to the protocol of Gregan et al.47 (see mendel.imp.ac.at/Pombe_deletion). Haploid meiosis was analyzed as described in reference 31. Construction of lys1-GFP and cen2-GFP constructs was described in references 48 and 32 respectively. The genotypes of S. pombe strains used in this study are listed in the Table 1. Acknowledgements
We thank F. Klein, J. Loidl, A. Amon, V. Katis, C. Rumpf, A. Yamamoto and Y. Watanabe for helpful discussions and K. Gull for the TAT1 antibody. We thank S. Westermann and J.M. Peters for allowing us to use the tetrad dissection
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Table 1. Strain list Figure
Strain number
Genotype
Figure 1A
JG12771
h mat-M cdc2-L7 LacO-lys1+ GFP-LacI-his7+
Figure 1A
JG12863
h+ mat-M cdc2-L7 LacO-lys1+ GFP-LacI-his7+ sgo1::natMX
Figure 1A
JG14611
h+ mat-M cdc2-L7 LacO-lys1+ GFP-LacI-his7+ par1::kanMX
Figure 1B
JG11338
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210
Figure 1B
JG11339
h+ lys1 his7 leu1 ura4 ade6-210
Figure 1B
JG12226
h leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP
Figure 1B
JG11339
h+ lys1 his7 leu1 ura4 ade6-210
Figure 1B
JG11792
h LacO-lys1 GFP-LacI-his7+ leu1 ura4 ade6-210 sgo1::natMX
Figure 1B
JG11793
h+ lys1 his7 leu1 ura4 ade6-210 sgo1::natMX
Figure 1B
JG12269
h- leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP sgo1::natMX
Figure 1B
JG12340
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 rec12::kanMX
Figure 1B
JG12342
h+ lys1 his7 leu1 ura4 ade6-210 rec12::kanMX
Figure 1B
JG14860
h cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP rec12::kanMX
Figure 1B
JG11798
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 mde2::natMX
Figure 1B
JG11799
h+ lys1 his7 leu1 ura4 ade6-210 mde2::natMX
Figure 1B
JG14952
h leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP mde2::natMX
Figure 1B
JG12986
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 rec12::kanMX sgo1::natMX
Figure 1B
JG12987
h+ lys1 his7 leu1 ura4 ade6-210 rec12::kanMX sgo1::natMX
Figure 1B
JG14957
h- cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP rec12::kanMXsgo1::hygMX
Figure 1B
JG14958
h+ lys1 his7 leu1 ura4 ade6-210 mde2::natMX sgo1::hygMX
Figure 1B
JG14959
h- leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP mde2::natMXsgo1::hygMX
Figure 1B
JG13988
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 ppa2::kanMX rec12::hygMX
Figure 1B
JG13989
h+ lys1 his7 leu1 ura4 ade6-210 ppa2::kanMX rec12::hygMX
Figure 1B
JG12828
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 ppa2::kanMX
Figure 1C
JG11338
h- LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210
Figure 1C
JG11339
h+ lys1 his7 leu1 ura4 ade6-210
Figure 1C
JG12226
h leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP
Figure 1C
JG14957
h- cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP rec12::kanMXsgo1::hygMX
Figure 1C
JG12987
h+ lys1 his7 leu1 ura4 ade6-210 rec12::kanMX sgo1::natMX
Figure 1C
JG13988
h LacO-lys1+ GFP-LacI-his7+ leu1 ura4 ade6-210 ppa2::kanMX rec12::hygMX
Figure 1C
JG13989
h+ lys1 his7 leu1 ura4 ade6-210 ppa2::kanMX rec12::hygMX
Figure 2
JG12226
h leu1-32 lys1-131 ura4-D18 cen2(D107)::Kan-ura4+-lacO his7+::lacI-GFP
Figure 2
JG11339
h+ lys1 his7 leu1 ura4 ade6-210
Figure 2
JG14860
h leu1-32 lys1-131 ura4-D18 cen2(D107)::kan-ura4+-lacO his7+::lacI-GFP rec12::kanMX
Figure 2
JG12342
h+ lys1 his7 leu1 ura4 ade6-210 rec12::kanMX
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-
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-
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Institute, Laboratory of Molecular Genetics, 83391, Bratislava, Slovak Republic, supported by the FWF Lise Meitner Program M1145.
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