E Average dissociation frequencies of sister centromeres in wild-type and rec12 mutant cells. The number of centromeres examined is shown in parentheses. The stage was determined based on centromere behavior and the distance between the spindle poles visualized using the DsRed-tagged SPB component Sad1 not shown. Arrowheads indicate sister centromeres that underwent dissociation. Numbers at the top indicate the time in seconds. In analyses of centromere position and dissociation, 20 and 28 pairs of sister centromeres were examined for wild-type and rec12 mutant strains, respectively.
More than 10 time points were examined for each centromere analysis. Notably, we found that sister centromeres occasionally underwent a transient dissociation in both wild-type and rec12 mutant cells Figure 4A and 4D , Table 1.
This dissociation was not the result of the integration into the chromosome of lacO repeats, which are used for visualization [33] , or of the dissociation of only the visualized pericentromeric region; when all three homologous sets of sister centromeres were visualized by GFP tagging of the centromere-specific histone H3 variant Cnp1 [43] , we observed more than six centromere signals together with a transient split of the signal into two Figure 4F.
These observations showed that bipolar attachment of sister chromatids occasionally occurs during the pre-anaphase stage, irrespective of chiasma formation. Similar centromere dynamics were also observed in cells lacking Sgo1. The occurrence of bipolar attachment in the presence of chiasmata is contradictory to the idea that chiasmata prevent the bipolar attachment of sister chromatids from occurring during the pre-anaphase stage.
If chiasmata do not prevent the bipolar attachment of sister chromatids from occurring, they must contribute to the elimination of bipolar attachment of sister chromatids during the pre-anaphase stage. However, the overall frequency of centromere dissociation was not significantly different between wild-type and rec12 mutant cells Figure 4E , Table 1 , and chiasma-dependent elimination of the bipolar attachment was not evident.
We hypothesized that if sister centromeres attach to both poles more frequently in the achiasmate background, the chiasma-dependent elimination of the bipolar attachment would be evident. Following this hypothesis, we examined mrc1 and moa1 mutant cells. The mrc1 gene encodes a conserved DNA replication checkpoint factor, which delays cell cycle progression upon DNA replication stress, promotes proper fork progression, and contributes to sister chromatid cohesion in mitosis [44] — [50].
On the other hand, the moa1 gene encodes a meiosis-specific centromere protein that contributes to the proper centromere localization of the meiotic cohesin component Rec8 [34]. In both mrc1 and moa1 mutant cells, chromosome segregation as well as spindle dynamics, recombination, and spore formation are largely normal Figures S2B and S3 , Text S1 [34]. Although these phenotypes are similar to the sgo1 -mutant phenotypes, the equational segregation is primarily caused by defects in centromere features other than maintenance of centromere cohesion, because both mrc1 and moa1 mutant cells can maintain sister centromere cohesion until anaphase II if sister chromatids are not segregated equationally during meiosis I Figure S3D , Text S1 [34].
Therefore, the equational segregation seen in the mrc1 rec12 and moa1 rec12 mutant cells is likely to be caused by frequent bipolar attachment of sister chromatids, and we expected that the chiasma effects would be more evident in the mrc1 and moa1 mutants. A Sister chromatid segregation in mrc1 and moa1 mutants and the effects of Rec12 or Mad2 depletion analyzed by the GFP-visualized cen2. B Sister chromatid segregation in moa1 mutant and the effects of Sgo1 or Rec12 depletion analyzed by the GFP-visualized cen1.
C Effects of Mrc1 or Moa1 depletion on sister chromatid segregation at meiosis I in haploid cells. Sister chromatid segregation was analyzed by the GFP-visualized cen2. Data values in all graphs were obtained as described in Figure 2. Asterisks show statistically significant differences and their associated p values. To evaluate chiasma effects in the mrc1 and moa1 mutants, we first examined the pre-anaphase centromere dynamics in the achiasmate mrc1 rec12 and moa1 rec12 double-mutant cells.
In the mrc1 rec12 mutant cells, the sister centromeres dissociated more frequently Figure 6A and 6B , with a significantly longer duration Table 1 , and were predominantly positioned around the spindle center, unlike those in the rec12 mutant cells Figure 6C.
In the moa1 rec12 mutant cells, the centromeres were also frequently positioned around the spindle center Figure 6A and 6C , and in addition, the SAC was not activated as much as in rec12 mutant cells Figure S2A , Text S1. These characteristics were expected to be associated with frequent bipolar attachment of sister chromatids Figure 6D.
Indeed, the frequent dissociation of the centromeres and their positioning around the spindle center together with the low level of SAC activation were observed during meiosis I in achiasmate rec8 mutant cells Figure 6A—6C and Figure S2A , Table 1 , in which sister chromatids efficiently attach to both poles to fully undergo equational segregation [12] , [51]. They were also observed during mitotic division in wild-type diploid cells Figure S4.
These observations thus confirmed that sister centromeres attach to both poles more frequently in the mrc1 rec12 and moa1 rec12 double-mutant cells than in rec12 single-mutant cells. However, the centromere properties of the mrc1 and moa1 mutant cells differed from those of rec8 mutant or mitotic cells because the SAC substantially delayed anaphase initiation in mrc1 rec12 mutant cells Figure S2A , Text S1 , and centromere dissociation was not so frequent in moa1 rec12 mutant cells Figure 6B.
A Pre-anaphase dynamics of the spindle pole and centromere cen2 at meiosis I, and changes in the distance between the spindle pole and the centromere and between the two spindle poles in mrc1 , moa1 , and rec8 mutants. Note that only one of the homologous centromeres is visualized in mrc1 rec12 and rec8 mutant cells. B Average centromere dissociation frequencies in mrc1 , moa1 , and rec8 mutant cells. Asterisks indicate dissociation frequencies that are statistically different from the frequency of wild type.
C Observation frequencies of centromeres at distinct positions in the spindle during the pre-anaphase stage. The positions of centromeres are shown based on their relative distance from the spindle center d , as determined in Figure 4B.
D Bipolar attachment of sister chromatids and expected observation frequencies of centromeres at distinct positions in the spindle. E Distance between homologous centromeres. The distance between homologous centromeres was measured at every time point in each strain, and an average distance is shown.
When centromeres were dissociated, the distance between the nearest homologous pair of centromeres was measured. The number of distances examined is shown in parentheses.
Right illustrations show models for spindle attachment of chromosomes and the resultant distance between the centromeres in wild-type, mrc1 , and moa1 mutant cells. White arrows in all illustrations indicate forces exerted on chromosomes. Error bars in all graphs indicate standard deviations. We next examined the pre-anaphase centromere dynamics in the chiasmate mrc1 and moa1 single-mutant cells to evaluate chiasma effects.
Remarkably, in mrc1 single-mutant cells, the level of centromere dissociation was almost identical to that in wild-type cells Figure 6A and 6B , Table 1 , indicating that bipolar attachment of sister chromatids was reduced to a wild-type level.
Furthermore, centromere positioning and the distance between homologous centromeres were very similar to what was seen in wild-type cells Figure 6C and 6E , indicating that homologous chromosomes attach to both poles as frequently as in wild-type cells.
These results show that chiasmata eliminate the bipolar attachment of sister chromatids and promote the bipolar attachment of homologous chromosomes during the pre-anaphase stage in mrc1 mutant cells.
On the other hand, in moa1 mutant cells, centromere positioning and dissociation were not significantly different from those seen in achiasmate moa1 rec12 mutant cells Figure 6A—6C , Table 1. Furthermore, homologous centromeres were not separated as widely as in wild-type cells Figure 6E. These results indicate that sister chromatids still attach to both poles at a level similar to that in moa1 rec12 mutant cells and pulling forces are not properly exerted on homologous chromosomes in moa1 mutant cells Figure 6E.
Therefore, chiasmata fail to eliminate the bipolar attachment of sister chromatids during the pre-anaphase stage in moa1 mutant cells. Because the bipolar attachment of sister centromeres did not appear to be eliminated during the pre-anaphase stage in chiasmate moa1 mutant cells, we examined whether their bipolar attachment is retained during anaphase by analyzing anaphase centromere dynamics. In wild-type cells, sister centromeres moved swiftly toward the poles all 13 of the centromeres examined reached the poles within s; Figure 7 and only occasionally dissociated during anaphase I [only three centromeres out of 13 The centromeres also moved swiftly to the pole and remained associated in mrc1 mutant cells all 11 centromeres examined reached the pole within 80 s without dissociation; Figure 7.
In contrast, in moa1 mutant cells, lagging and dissociation of centromeres were frequently observed during anaphase [10 out of 14 centromeres Furthermore, elimination of anaphase centromere cohesion by Sgo1 deletion substantially increased the equational segregation of sister chromatids Figure 5B.
These results showed that sister chromatids were frequently attached to both poles and pulled from opposite directions during anaphase I in moa1 mutant cells.
Surprisingly, most of the lagging centromeres eventually moved to the proper pole Figure 5A and 5B , Figure 7. Therefore, the chiasma generates a bias toward the proper pole in poleward chromosome pulling from opposite directions that eventually results in proper chromosome segregation in moa1 mutant cells.
Arrows and arrowheads show each of the homologous centromeres cen2 , respectively, and the two arrowheads or arrows indicate dissociated sister centromeres. In the current study, we examined the role of chiasmata by analyzing the segregation and dynamics of chromosomes during meiosis I induced in recombination-deficient diploid cells and in haploid cells.
The analysis of these two distinct types of achiasmate cells provided two lines of evidence to show that sister chromatids frequently attach to both poles and experience pulling forces from opposite directions during anaphase I in achiasmate cells. Second, when sister centromere cohesion was resolved during anaphase by Sgo1 depletion, sister chromatids frequently underwent equational segregation during anaphase I Figure 2B and 2D. Chiasmata therefore play a crucial role in preventing the bipolar attachment of sister chromatids during anaphase I.
Because the bipolar attachment of sister chromatids has been observed during anaphase I in various achiasmate organisms [27] — [29] , it is probably common among eukaryotes.
We further examined how chiasmata prevent the bipolar attachment of sister chromatids. Loss of chiasmata causes activation of the SAC [42]. However, we showed that the bipolar attachment of sister chromatids depends only partially on the SAC in achiasmate cells. The reduction of the bipolar attachment that normally generates tension in the achiasmate background is consistent with the idea that the SAC promotes attachments that generate tension [40] , [41].
We performed high time-resolution analysis of pre-anaphase centromere dynamics in several different types of chiasmate and achiasmate cells to understand how chiasmata contribute to the attachment.
From this analysis, we have reached three conclusions. First, chiasmata cannot prevent occurrence of bipolar attachment of sister chromatids, based on the observation that the bipolar attachment occasionally occurred in chiasmate wild-type cells. Second, analysis of mrc1 mutant cells showed that chiasmata contribute to the elimination of the bipolar attachment of sister chromatids during the pre-anaphase stage Figure 8A.
However, the elimination was not evident in wild-type cells in comparison with rec12 mutant cells. One possible explanation for this result is that the bipolar attachments occur more frequently in wild-type than in rec12 mutant cells because the centromere is positioned closer to the spindle center in wild-type cells Figure 4B.
Alternatively, chiasmata may eliminate bipolar attachments in mrc1 mutant cells but not in wild-type cells because of distinct centromere structures or functions. Furthermore, we cannot completely exclude the possibility that the chiasmata-dependent elimination depends in part on unknown Rec12 functions.
A Chiasmata eliminate the bipolar attachment of sister centromeres centromeres on left sister chromatids during the pre-anaphase stage of meiosis I. B When the bipolar attachment remains during anaphase, chiasmata generate bias in the poleward pulling forces to cause proper chromosome segregation.
White arrows indicate the pulling forces exerted on chromosomes during anaphase I. A smaller arrow indicates a weaker or less continuously exerted force. For simplicity, only a single microtubule is shown to illustrate the spindle attachment of each kinetochore. Third, analysis of moa1 mutant cells showed that chiasmata induced a bias toward the proper pole in poleward chromosome pulling from opposite directions that resulted in proper chromosome segregation Figure 8B.
We also observed this chiasma effect, albeit occasionally, in wild-type cells Figure 7 , Wt, lower panel and thereby speculate that the chiasma-induced bias is a backup mechanism that ensures proper meiotic chromosome segregation even when improper attachments remain.
How the chiasmata eliminate bipolar attachments and induce a bias in chromosome pulling remains elusive. Because chiasmata are essential for generating the tension that stabilizes kinetochore—microtubule interactions and increases kinetochore microtubules [9] , [52] , we speculate that chiasmata execute these different tasks via tension, as follows see also Text S1.
In wild-type cells, sister kinetochores occasionally attach to both poles Figure S5A. In the presence of chiasmata, microtubules that attach to the proper poles generate sufficient tension, but those that attach to improper poles probably do not. As a result, improper attachments are eliminated while proper attachments are increased. In contrast, improper attachments are not eliminated in rec12 mutant cells, possibly because the improper attachments also generate tension Figure S5A.
In this model, chiasmata must prevent improper attachments from generating tension. During the pre-anaphase stage, chromosomes oscillate between the poles, and oscillation of the chiasma-linked chromosomes may reduce tension Figure S5B. When a pair of sister chromatids follows the other homologous pair that is moving toward the spindle pole, the leading sister chromatid pair presumably exerts pulling forces on the chromosome arms of the following pair via chiasmata.
These pulling forces are likely to reduce the tension that improper attachments generate but not those generated by proper attachments. As a result, only proper attachments i.
Alternatively, the chiasmata-dependent pulling may make the kinetochores on the following chromosomes face the side opposite the direction of chromosome movement to physically eliminate improper attachments. Although the above model can account for the observed chiasmata-dependent effects, we cannot completely rule out the possibility that chiasmata directly contribute to centromere function or structure to affect spindle attachment and segregation of chromosomes.
Chiasmata eliminated bipolar attachment of sister chromatids in the mrc1 mutant but did not eliminate it in the moa1 mutant. Given the frequent monopolar attachment of sister chromatids in the chiasmate mrc1 single-mutant cells together with the substantial SAC activation in achiasmate mrc1 rec12 double-mutant cells, sister kinetochores probably face the same side in mrc1 mutants.
However, the frequent bipolar attachment of sister chromatids seen in mrc1 rec12 mutant cells conversely implies that the kinetochores face opposite sides.
This contradiction may be explained by the flexibility of the kinetochore arrangement Figure S5A , Text S1. It is possible that in the mrc1 mutant cells, although sister kinetochores are initially arranged side by side, the kinetochores end up facing opposite sides when they are pulled from opposite directions, leading to the subsequent efficient bipolar attachment of sister centromeres.
On the other hand, in moa1 mutant cells, sister kinetochores perhaps face opposite sides to attach to both poles efficiently Figure S5A , Text S1 , as proposed previously [34]. Although kinetochore arrangement was previously proposed to be flexible in moa1 mutant cells [34] , we speculate that the arrangement is conversely inflexible because of strong centromere cohesion, considering increased centromere accumulation of cohesin [34] , infrequent sister centromere dissociation Figure 6B , and a narrower dissociation distance Figure S6.
Bipolar attachment was not eliminated in moa1 single-mutant cells, perhaps because bipolar attachment is easily re-established due to the back-to-back kinetochore arrangement.
An alternative possibility is that moa1 mutant cells are defective in destabilizing the kinetochore—microtubule interaction and fail to eliminate improper attachments efficiently. Our findings have three important implications for understanding the mitotic chromosome segregation mechanism. First, the frequent bipolar attachment of sister chromatids seen in achiasmate cells indicates that kinetochore arrangement alone cannot prevent improper attachments and suggests that bipolar merotelic attachment of a single chromatid also occurs when sister chromatid cohesion is defective.
Indeed, Courtheoux et al. Furthermore, a lagging chromatid was frequently observed during anaphase II in sgo1 mutant of fission yeast, in which sister chromatids undergo precautious dissociation before anaphase II [17]. These observations may alter the interpretation of phenotypes associated with monopolin and heterochromatin mutants of fission yeast, which were proposed to be defective in the arrangement of microtubule-binding sites of kinetochores because these mutants frequently exhibited merotelic attachments during mitotic anaphase [54] , [55].
However, defective sister centromere cohesion in the monopolin and heterochromatin mutants may have caused the merotelic attachments [56] — [58].
Second, the fact that sister chromatids, despite their bipolar attachment, move to the same pole in chiasmate cells indicates that monopolar attachment of sister chromatids is not a prerequisite for their proper segregation. This feature is probably common during mitotic chromosome segregation because the proper segregation of a single chromatid that is attached to both poles has also been observed in higher eukaryotes during mitosis [59].
Therefore, generation of bias in the segregation forces is probably a general mechanism that ensures correct chromosome segregation. Finally, the chromosome oscillation-dependent model for the elimination of improper attachments may also account for the establishment of proper attachments during mitosis Figure S5B.
During mitosis, chromosomes oscillate during the establishment of their spindle attachment Figure S4A [60] , [61] , and merotelic attachment occurs in higher eukaryotes [62]. Furthermore, in fission yeast, the physical linkage between two kinetochores induces their bipolar attachment during mitosis [63]. These facts suggest that the oscillation of cohesin-linked sister chromatids destabilizes improper attachments and contributes to the selection of proper attachments during mitosis.
In summary, we have shown that chiasmata are essential for proper spindle attachment and segregation of sister chromatids during meiosis I. Based on our results, we propose that chiasmata play a pivotal role in the selection of proper attachments and establish a backup mechanism that promotes the appropriate segregation of chromosomes when improper attachments remain during anaphase I.
Furthermore, we propose a model to explain how chromosome association contributes to correct spindle attachment of the chromosomes not only in meiosis but also in mitosis. Table S3 lists the yeast strains used in this study, and strains used in figures are described in Text S1. Media used in this study have been described by Moreno et al. For the segregation analyses of homologous chromosomes, two types of cells, both of which contained GFP-labeled centromeres cen2 or lys1 , were crossed on solid ME medium.
For sister chromatid segregation analyses, cells containing GFP-labeled centromeres were crossed with cells lacking GFP-labeled centromeres. Zygotes containing two DNA masses with a tear-drop shape and pointed ends facing each other were excluded because they were in the karyogamy stage. Haploid yeast cells were forced to enter meiosis by Pat1 inactivation following activation of the mating pheromone signaling pathway, as previously described [33]. Meiotic progression was monitored by analysis of chromosomal DNA morphology at 1-h time intervals.
Sister chromatid segregation was analyzed in cells containing two DNA masses that underwent meiosis I. The behavior of the GFP-labeled chromosome locus was observed every 1 min or 10 s. A set of images from six focal planes with 0.
All measurements were conducted in three dimensions. Chromosome dynamics at meiosis I in haploid cells. Magenta and green show chromosomes and the spindle, respectively. Numbers indicate time in minutes. Arrowheads indicate three chromosomes, and arrows indicate lagging chromosomes during anaphase I. Anaphase initiation timing and spindle dynamics in various types of cells. A Timing of anaphase I onset examined by phase II duration of spindle elongation.
Phase II duration of spindle elongation was examined, as previously reported [42]. The illustration shows typical elongation of the meiosis-I spindle over time and three phases in spindle elongation. Error bars show standard deviation. Values of spindle duration in Wt, mad2 , rec12 , and mad2 rec12 cells were adopted from our previous manuscript [42].
The number of spindles examined is shown in parentheses. Asterisks show durations that are statistically different from the duration of wild type and their associated p values, as determined by t-tests. B Spindle dynamics. Photos show spindle dynamics in mrc1 rec12 and moa1 rec12 mutants. Each graph shows changes in the length of 6 spindles.
Spore viability and chromosome segregation in mrc1 mutant. A Number of spores formed in wild-type and mrc1 asci. Graph shows average percentages obtained from two independent experiments. More than asci were examined for each strain in each experiment.
B Average spore viability of wild type and mrc1 mutant. Four spores in wild-type and mrc1 asci were dissected and examined for their viability by colony formation. Average spore viabilities of wild type and mrc1 mutant were obtained respectively from 4 and 7 independent experiments. At least 10 asci were dissected in each experiment. C Meiotic segregation of both homologous chromosomes and sister chromatids. Cells containing GFP-visualized cen2 of both homologous chromosomes were induced to meiosis, and chromosome segregation was examined in four nuclear cells that completed two divisions.
Bars show percentages of cells containing four nuclei, each of which contains a single GFP signal. D Sister chromatid segregation at meiosis II. Segregation of sister chromatids at meiosis II was examined by segregation patterns of GFP-visualized cen2 of one of the homologous chromosomes in four nuclear cells, and bars show average percentages of sister chromatid disjunction black and non-disjunction white at meiosis II. Cells which segregated sister chromatids equationally at meiosis I were excluded.
The percentages in wild-type, mrc1 , and sgo1 mutant cells were obtained from 2, 4, and 3 independent experiments, respectively, and more than 40 zygotic cells were examined in each experiment. Error bars in B and D indicate standard deviation.
Centromere dynamics during the mitotic pre-anaphase stage in diploid wild-type cells. A Pre-anaphase dynamics of the spindle pole and centromere cen2. B Average dissociation frequencies and duration of sister centromeres.
At least 10 consecutive time points were examined for each analysis. The number of examined positions is shown in parenthesis. Models for spindle attachment in various mutants and chromosome oscillation-dependent elimination of improper attachments. A Sister kinetochore arrangement and changes in spindle attachment of the kinetochores after their attachment to opposite poles Bipolar attachment. Tension generation: generation of tension white arrows on sister kinetochores by microtubules.
Selection: elimination of kinetochore-interacting microtubules. Amplification: increase of kinetochore-interacting microtubules. B Chromosome oscillation model for selection of proper attachments. Bipolar attachments of sister centromeres Meiosis I, centromeres on left sister chromatids or a single centromere Mitosis, a left centromere occasionally occurs, and tension white arrows is generated at the centromeres Improper attachment.
Movement of chiasma- or cohesin-linked chromosomes eliminates tension generated at the sites of improper attachments at meiosis I or in mitosis, respectively Loss of tension , because the leading chromosome s exerts pulling forces small gray arrows on the following chromosome s at the chromosome linkage sites.
Elimination of tension leads to detachment of improperly interacting microtubules Detachment. Large gray arrows in A and B indicate chromosome movement. Average distance between dissociated sister centromeres. The number of examined distances is the following. Wt: ; rec12 : ; mrc1 : ; mrc1 rec12 : ; moa1 : ; moa1 rec12 : ; rec8 : In moa1 mutants, the distance between dissociated sister centromeres was significantly shorter than in wild-type cells.
Strain list [67] — [74]. Supplementary text [66]. We also thank Koichi Tanaka and Yoshinori Watanabe for sharing unpublished results and Kayoko Tanaka, Masahiro Uritani, Takashi Ushimaru, and Takanori Oyoshi for their critical reading of our manuscript and helpful comments. The authors have declared that no competing interests exist.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. PLoS Genet.
Published online Mar Gregory P. Copenhaver, Editor. Author information Article notes Copyright and License information Disclaimer. Received Aug 18; Accepted Feb 8. Copyright Hirose et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
This article has been cited by other articles in PMC. Figure S2: Anaphase initiation timing and spindle dynamics in various types of cells.
Figure S3: Spore viability and chromosome segregation in mrc1 mutant. Figure S4: Centromere dynamics during the mitotic pre-anaphase stage in diploid wild-type cells. Figure S5: Models for spindle attachment in various mutants and chromosome oscillation-dependent elimination of improper attachments. Figure S6: Average distance between dissociated sister centromeres. Table S1: Parameters of centromere movements during the pre-anaphase stage at meiosis I.
Table S2: Direction of centromere movements during the pre-anaphase stage at meiosis I. Table S3: Strain list [67] — [74]. Text S1: Supplementary text [66]. Abstract The chiasma is a structure that forms between a pair of homologous chromosomes by crossover recombination and physically links the homologous chromosomes during meiosis.
Author Summary Gametes form through a special type of cell division called meiosis. Introduction During cell division, chromosomes that harbor genetic information are accurately segregated into daughter cells.
Open in a separate window. Figure 1. Spindle attachment of chromosomes and their segregation during mitosis and meiosis I. Results Sister centromeres frequently become dissociated and remain between the spindle poles during anaphase I in rec12 mutant cells Elimination of chiasmata induced by depletion of Rec12, a recombination factor required for the formation of double-strand breaks [36] , causes occasional equational segregation of sister chromatids [33] and frequent non-disjunction of homologous chromosomes [37].
Figure 2. The effect of loss of chiasmata on chromosome segregation. Sgo1 depletion causes equational segregation of sister chromatids during meiosis I in achiasmate cells To confirm the frequent bipolar attachment of sister chromatids in achiasmate cells, we depleted Sgo1, which inhibits the removal of centromeric cohesin during anaphase I [17] , [19]. Bipolar attachment of sister chromatids only partially depends on the spindle assembly checkpoint SAC in achiasmate cells The SAC ensures faithful chromosome segregation by delaying anaphase initiation until all of the chromosomes become properly attached to the spindle [40] , [41].
Figure 3. Bipolar attachment of sister chromatids occasionally occurs before anaphase irrespective of chiasma formation Spindle attachment of chromosomes is established before anaphase, and the chiasma may prevent the bipolar attachment of sister chromatids from occurring during the pre-anaphase stage.
Figure 4. Pre-anaphase centromere dynamics during meiosis I in wild-type and rec12 mutant cells. Table 1 Centromere dissociation during the pre-anaphase stage of meiosis I in various fission yeast strains.
Sister chromatids attach to both poles more frequently in mrc1 rec12 and moa1 rec12 double-mutant cells than in rec12 single-mutant cells If chiasmata do not prevent the bipolar attachment of sister chromatids from occurring, they must contribute to the elimination of bipolar attachment of sister chromatids during the pre-anaphase stage. Figure 5. Sister chromatid segregation in mrc1 and moa1 mutants. Figure 6. Pre-anaphase centromere dynamics during meiosis I in mrc1 and moa1 mutants.
Chiasmata prevent the bipolar attachment of sister chromatids in mrc1 mutant cells but not in moa1 mutant cells We next examined the pre-anaphase centromere dynamics in the chiasmate mrc1 and moa1 single-mutant cells to evaluate chiasma effects. Chiasmata induce the preferential exertion of segregation forces on sister chromatids toward the proper pole during anaphase I in moa1 mutant cells Because the bipolar attachment of sister centromeres did not appear to be eliminated during the pre-anaphase stage in chiasmate moa1 mutant cells, we examined whether their bipolar attachment is retained during anaphase by analyzing anaphase centromere dynamics.
Figure 7. Centromere dynamics during anaphase I in mrc1 and moa1 mutants. Discussion Chiasmata play a crucial role in preventing the bipolar attachment of sister chromatids during anaphase I In the current study, we examined the role of chiasmata by analyzing the segregation and dynamics of chromosomes during meiosis I induced in recombination-deficient diploid cells and in haploid cells.
Two distinct tasks of chiasmata: elimination of the bipolar attachment of sister chromatids and induction of a bias in poleward chromosome pulling We further examined how chiasmata prevent the bipolar attachment of sister chromatids.
Figure 8. Two major roles of chiasmata during meiosis I. Mechanism associated with chiasma-dependent elimination of bipolar attachments and biased chromosome pulling How the chiasmata eliminate bipolar attachments and induce a bias in chromosome pulling remains elusive.
Effects of kinetochore arrangement on the chiasma-dependent elimination of improper attachments Chiasmata eliminated bipolar attachment of sister chromatids in the mrc1 mutant but did not eliminate it in the moa1 mutant. Common mechanisms for chromosome segregation between mitosis and meiosis Our findings have three important implications for understanding the mitotic chromosome segregation mechanism. Materials and Methods Yeast strains and media Table S3 lists the yeast strains used in this study, and strains used in figures are described in Text S1.
Analysis of chromosome segregation during meiosis I in haploid cells Haploid yeast cells were forced to enter meiosis by Pat1 inactivation following activation of the mating pheromone signaling pathway, as previously described [33]. In addition, heterozygous regions increase chiasma formation when are juxtaposed with homozygous regions, which reciprocally decrease Ziolkowski et al. In relation to chromosome 3, Barth et al. In general, there are difficulties in mapping and sequencing this chromosome, consequently this fact suggests the existence of unusual chromatin-related features respect to the other chromosomes of the complement Schmidt, Taking into account the information compiled in this work, it is evident that when the chromosome complement of a diploid individual is duplicated, the degree of relationship between two chromosomes within each tetrasome may be greater than mere homology.
In this situation, there is a competition for chiasma formation between identical and homologous chromosomes that can be resolved through different ways depending on the chromosome. Accordingly, identical and homologous chromosome regions will persist in each tetrasome in a differential pattern throughout generations. This chromosomal genetic variation has not be considered in current models about tetrasomic and disomic inheritance and it could produce a relevant impact on haplotypes.
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