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. Choose the variations of the temperature at which you want to experiment and study the relation.
Maintain the insects at the chosen temperature. It is recommended to use more insects to eliminate all possible differences during the experiment. Recombination and the frequency of recombinants The recombination is a process in which the DNA strands break and recombine to form a new combination of alleles, called recombinant. The estimation of both, frequency of recombination and the chiasmata frequency, is required in the construction of accurate genetic or linkage maps and to determine the whole length of the genome.
Method for the estimation of the chiasmata frequency in the arabidopsis A study by Moran et. Slide Preparation Wash the fixed buds in a fixative consisting of ethanol and glacial acetic acid in the ratio followed by citrate buffer pH4.
Incubate the buds in an enzyme mixture consisting of, 0. Put the buds in an ice-cold buffer to stop the reaction. Transfer a single bud to a clean slide with a small volume of buffer. Macerate the bud on the slide using a fine needle. Rinse off the fixative from the slide and dry it using a hairdryer. Statistical analyses Analyze the chiasmata data using the Minitab software and note down your results.
Method for the estimation of the chiasmata frequency in the Mice This section presents a study by Gorlov et al. Procedure Prepare and characterize the somatic karyotypes of the organism on metaphases from bone marrow biopsy.
Take the testicular material from the adult mice for meiotic preparations. Prepare diplotene-diakinesis chromosome spreads by treating the sample with a hypotonic solution. After the treatment, fixation is done using the solution of methanol and acetic acid prepared in a ratio. Process the slides with the C-banding staining technique. Observe and analyze the spread of diplotene-diakinesis using light microscopy to locate chiasmata and determine the chiasma frequency by the chi-square method 2.
What characteristics should you observe while noting down your results? Here are a few points: Measure the relative length and centromere index of individual chromosomes. Analyze the distribution of the chiasma between cells. Study the correlation between chiasma frequency per cell and total autosomal length. Observe if there is competition for chiasmata between different chromosomes of the same cell.
Calculate the number of chiasmata formed on individual chromosomes and chromosome arms. Analyze the effect of chiasma interference over the centromere. Find the relation between the mean chiasma frequency and chromosomes as well as chromosome arm length and calculate the correlation coefficient statistical term to determine the relationship between two variables.
Study and analyze the distribution of the chiasmata within chromosomes arms. You can draw a histogram to analyze the results efficiently. Conclusion Genetic recombination is the main source of variability in a population, which is necessary for the continued evolution. References Cano, M. The relationship between chiasma frequency and bivalent length: Effects of genotype and supernumerary chromosomes.
Heredity , 56 3 , — Pradillo, C. Oliver, C. Romero, N. Santos Looking for natural variation in chiasma frequency in Arabidopsis thaliana. Sanchez-Moran, S.
Armstrong, J. Santos, F. Franklin, and G. Jones Genetics 3 , Grieco, M. Chiasma frequency and distribution in males and females of Metaleptia brevicornis adspersa Acridinae, Acrididae with and without B chromosomes. Hereditus, , — Lund, Sweden.
ISSN Henderson, S. Chiasma distribution at diplotene in a locust. Heredity , 18 2 , — Hulten M. Given these two mechanisms, it is highly unlikely that any two haploid cells resulting from meiosis will have the same genetic composition Figure 3. Figure 3. In this case, there are two possible arrangements at the equatorial plane in metaphase I. The total possible number of different gametes is 2 n , where n equals the number of chromosomes in a set.
In this example, there are four possible genetic combinations for the gametes. To summarize the genetic consequences of meiosis I, the maternal and paternal genes are recombined by crossover events that occur between each homologous pair during prophase I.
In addition, the random assortment of tetrads on the metaphase plate produces a unique combination of maternal and paternal chromosomes that will make their way into the gametes. In anaphase I, the microtubules pull the linked chromosomes apart. The sister chromatids remain tightly bound together at the centromere.
The chiasmata are broken in anaphase I as the microtubules attached to the fused kinetochores pull the homologous chromosomes apart Figure 4. Figure 4. The process of chromosome alignment differs between meiosis I and meiosis II. In prometaphase I, microtubules attach to the fused kinetochores of homologous chromosomes, and the homologous chromosomes are arranged at the midpoint of the cell in metaphase I.
In anaphase I, the homologous chromosomes are separated. In prometaphase II, microtubules attach to the kinetochores of sister chromatids, and the sister chromatids are arranged at the midpoint of the cells in metaphase II.
In anaphase II, the sister chromatids are separated. In telophase, the separated chromosomes arrive at opposite poles.
The remainder of the typical telophase events may or may not occur, depending on the species. In some organisms, the chromosomes decondense and nuclear envelopes form around the chromatids in telophase I.
In other organisms, cytokinesis—the physical separation of the cytoplasmic components into two daughter cells—occurs without reformation of the nuclei. In nearly all species of animals and some fungi, cytokinesis separates the cell contents via a cleavage furrow constriction of the actin ring that leads to cytoplasmic division.
In plants, a cell plate is formed during cell cytokinesis by Golgi vesicles fusing at the metaphase plate. This cell plate will ultimately lead to the formation of cell walls that separate the two daughter cells. Two haploid cells are the end result of the first meiotic division.
The cells are haploid because at each pole, there is just one of each pair of the homologous chromosomes. Therefore, only one full set of the chromosomes is present. This is why the cells are considered haploid—there is only one chromosome set, even though each homolog still consists of two sister chromatids.
Recall that sister chromatids are merely duplicates of one of the two homologous chromosomes except for changes that occurred during crossing over. In meiosis II, these two sister chromatids will separate, creating four haploid daughter cells.
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