Climate Change 5: Evolution 1. Evolution Evidence 2. Natural Selection 3. Classification 4. Cladistics 6: Human Physiology 1.
Digestion 2. The Blood System 3. Disease Defences 4. Gas Exchange 5. Homeostasis Higher Level 7: Nucleic Acids 1. DNA Structure 2. Transcription 3. Translation 8: Metabolism 1. Metabolism 2. Cell Respiration 3. Photosynthesis 9: Plant Biology 1. Xylem Transport 2. Phloem Transport 3. Furthermore, shugoshin proteins maintain centromeric cohesion during anaphase I [17] — [21].
These proteins inhibit the removal of centromeric cohesin and regulate centromeric aurora kinase [17] — [19] , [21] — [24]. Elimination of both of these functions compromises sister chromatid segregation during meiosis I and II [17] , [18] , [22] , [25]. Further, elimination of the cohesin-retention function alone causes sister chromatid separation after anaphase I but has little if any effect on sister chromatid segregation toward the same pole during anaphase I [17] , [19].
Unlike the situation in mitosis, homologous chromosome association contributes to the generation of tension at the kinetochore in meiosis. Homologous chromosomes are physically associated with each other via the chiasmata that are formed by reciprocal recombination.
When homologous chromosomes are pulled in opposite directions, the chiasmata generate tension at the kinetochore and stabilize the kinetochore—microtubule interaction. Elimination of chiasmata leads to non-disjunction of homologous chromosomes [26]. In addition to this widely accepted role, chiasmata appear to play additional roles in the attachment of chromosomes to the spindle.
A lack of chiasmata results in the separation or fragmentation of sister chromatids during meiosis I in many species [27] — [29] , suggesting that chiasmata prevent the bipolar attachment of sister chromatids. Furthermore, chiasmata greatly alter meiotic sister chromatid segregation patterns in several different types of fission yeast cells.
Fission yeast cells normally undergo meiosis after responding to the mating pheromone [30] , but meiosis can also be induced without mating pheromone response by inactivation of Pat1 kinase, a key negative regulator of meiosis [31] , [32]. We previously reported that when haploid fission yeast cells lacking homologous chromosomes were forced to enter meiosis by Pat1 inactivation after a mating pheromone response, sister chromatids were primarily segregated to the same pole at meiosis I, as seen in normal diploid meiosis [33].
However, when they were induced to enter meiosis without a mating pheromone response, sister chromatids primarily underwent equational segregation.
By contrast, when Pat1 inactivation forced diploid cells to enter meiosis without a mating pheromone response, the sister chromatids were primarily segregated to the same pole in a recombination-dependent manner. Similar recombination-dependent co-segregation of sister chromatids has been observed in several cohesin-related mutants of fission yeast [34].
These findings suggest that chiasmata promote the monopolar attachment of sister chromatids; however, because a loss of recombination causes only a negligible level of equational segregation during normal diploid meiosis in fission yeast cells, chiasmata have previously been thought to be dispensable for monopolar attachment of sister chromatids [35]. The contribution of chiasmata to the monopolar attachment during meiosis I, therefore, remains elusive.
To understand the mechanisms underlying meiotic chromosome segregation, we examined the functions of chiasmata in spindle attachment and segregation of sister chromatids during meiosis I in fission yeast.
Our analysis of chromosome segregation and dynamics in several different types of achiasmate cells showed that in the absence of chiasmata, sister chromatids were frequently attached to opposite poles during anaphase I. High time-resolution analysis of centromere dynamics further showed that chiasmata contribute to the elimination of bipolar attachments during the pre-anaphase stage.
Furthermore, when the bipolar attachments remain during anaphase I, chiasmata induce a bias toward the proper pole during poleward chromosome pulling from opposite directions that results in correct chromosome segregation. Based on our findings, we discuss how chiasmata contribute to spindle attachment and segregation of chromosomes and further extend our idea to include the general functions of chromosome association during mitotic and meiotic chromosome segregation.
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].
As a first step toward understanding the role of chiasmata in the spindle attachment of sister chromatids, we re-examined chromosome segregation during meiosis I in more detail in rec12 mutant cells. We examined chromosome segregation by visualizing centromere-linked loci of chromosome I the lys1 locus: cen1 and chromosome II the D locus: cen2 using green fluorescent protein GFP [33]. After the first division, homologous centromeres were partitioned into two nuclei and rarely into the same nucleus in wild-type cells Figure 2A.
In contrast, homologous centromeres were frequently partitioned into the same nucleus in rec12 mutant cells Figure 2A. These results confirmed the mis-segregation of both homologous chromosomes and sister chromatids during meiosis I in rec12 mutant cells.
The same mis-segregation phenotypes were also observed in cells lacking the Rec14 recombination factor, which functions together with Rec12 and the depletion of which eliminates recombination Figure 2A and 2B [38] , [39]. A The frequency of non-disjunction of homologous chromosomes during meiosis I was examined by GFP-visualized cen2.
B Equational segregation of sister chromatids during meiosis I in various types of diploid cells. ND: not determined. C Centromere dynamics in wild-type and rec12 mutant cells during meiosis I.
Arrows indicate the spindle pole bodies SPBs. Arrowheads and barbed arrowheads show homologous centromeres cen2. Pre-anaphase: the pre-anaphase stage, as determined by a constant pole-to-pole distance. Anaphase I: anaphase I, as determined by an increase in the pole-to-pole distance. Numbers indicate the time in minutes from the beginning of spindle formation.
D Equational segregation of sister chromatids at meiosis I induced in haploid cells. Sister chromatid segregation was analyzed using GFP-visualized cen2. In all analyses in this study, with the exception of analyses in the supplementary results, sister chromatid segregation was analyzed in cells containing two DNA masses that underwent meiosis I.
Each data point was obtained from two independent experiments, with the exception of the non-disjunction frequency of homologous chromosome in rec12 mutant cells, which was obtained from three independent experiments.
More than 50 cells were examined in each experiment. Error bars indicate standard deviation. Asterisks indicate statistically significant differences and their associated p values, as determined by t-tests.
Segregation analysis showed that the overall mis-segregation frequency of sister chromatids in recombination-deficient, chiasmata-lacking cells i. However, live cell analysis of cen2 dynamics suggested that improper spindle attachment of sister centromeres occurs more frequently during anaphase I.
Although the sister centromeres eventually moved to the pole in rec12 mutant cells, they frequently remained between the two spindle poles and were dissociated during anaphase I [observed for 7 out of 14 centromeres examined The chromosome lagging is most likely caused by a loss of chiasmata and not a loss of Rec12 function, because lagging chromosomes were also frequently observed when meiosis was induced in haploid cells [33] , which do not form chiasmata due to their lack of homologous chromosomes Figure S1.
These results suggest that sister centromeres are frequently attached to both poles and are pulled from opposite directions during anaphase 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].
We hypothesized that although sister chromatids are frequently attached to both poles and are pulled from opposite directions, the centromere cohesion that persists until meiosis II should provide resistance against this force and prevent their separation during anaphase I in achiasmate cells. If so, depletion of Sgo1, which eliminates centromere cohesion during anaphase I, should lead to frequent equational segregation of sister chromatids.
Indeed, Sgo1 depletion led to a substantial increase in equational segregation in achiasmate cells. When meiosis I was induced in haploid cells, Sgo1 depletion similarly increased equational segregation Figure 2D irrespective of Rec12 depletion data not shown. Therefore, the increased equational segregation is not specific to recombination-deficient cells but is common in achiasmate cells.
These results confirm that the loss of chiasmata frequently leads to the bipolar attachment of sister chromatids during anaphase I. The SAC ensures faithful chromosome segregation by delaying anaphase initiation until all of the chromosomes become properly attached to the spindle [40] , [41].
We previously reported that the SAC becomes activated to delay anaphase initiation at meiosis I in rec12 mutant cells, which is likely associated with improper spindle attachment of chromosomes [42]. Similarly, analysis of spindle length showed that anaphase initiation was substantially delayed in sgo1 rec12 double-mutant cells in a Mad2-dependent manner, as previously observed in rec12 mutant cells Figure S2A , Text S1.
Therefore, we next examined whether the SAC contributes to the bipolar attachment of sister chromatids in achiasmate cells by depleting the SAC factor Mad2. Mad2 depletion led to decreased equational segregation of sister chromatids in rec12 mutant cells, but equational segregation was still observed at substantial levels in rec12 sgo1 double-mutant cells Figure 3A. Likewise, Mad2 depletion decreased but did not abolish equational segregation during meiosis I in haploid cells Figure 3B.
These results showed that the SAC promotes the bipolar attachment of sister chromatids but is not essential for this process in achiasmate cells. Furthermore, as seen in rec12 mutant cells, sister centromeres frequently dissociated and failed to move to the pole during anaphase I in mad2 rec12 double-mutant cells [ However, lagging centromeres were rarely observed in mad2 mutant cells [0.
These results indicated that the lagging centromeres seen in achiasmate cells were not caused by SAC activation or delayed anaphase initiation. Thus, we conclude that the bipolar attachment of sister chromatids depends only partially on the SAC in achiasmate cells. A Effects of Mad2 depletion on sister chromatid segregation during meiosis I in rec12 achiasmate cells. B Effects of Mad2 depletion on sister chromatid segregation at meiosis I in haploid cells. C Effects of Mad2 depletion on centromere dynamics during meiosis I.
Arrows indicate the SPBs. Arrowheads and barbed arrowheads show each of the homologous centromeres cen2. In A and B , each value was obtained from two independent experiments, with the exception of the equational frequencies for rec12 sgo1 mad2 cells, which were obtained from three independent experiments.
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.
To test this possibility, we examined the dynamics of sister centromeres before anaphase by time-lapse analysis with s intervals. The time-lapse analysis of cen2 loci on both homologous chromosomes in wild-type and rec12 mutant cells confirmed our previous observations from time-lapse analyses with 1-min intervals, although they exhibited slight differences in dynamic parameters Table S1 [42]. Homologous centromeres oscillated between the two spindle poles in a somewhat coordinated manner in wild-type cells; a pair of homologous centromeres often moved in the same direction Figure 4A , Table S2.
Accordingly, centromeres were mostly positioned around the middle point between the spindle pole and the spindle center with a tendency to be near the center Figure 4B. These centromere dynamics presumably reflect the frequent bipolar attachment of homologous chromosomes that are linked by the chiasmata Figure 4C.
On the other hand, sister centromeres oscillated in an uncoordinated manner and tended to remain near the pole in rec12 mutant cells Figure 4A , and centromere positioning was shifted toward the pole Figure 4B.
These centromere dynamics probably reflect the frequent attachment of each of the non-linked homologous chromosomes to one pole and the occasional switch in their attachment to the other pole Figure 4C. A Pre-anaphase dynamics of the spindle pole and centromere cen2 during meiosis I.
Photos were taken every 10 s and are shown in order from left to right. Horizontal bar: 50 s. B Observation frequencies of centromeres at distinct positions in the spindle during the pre-anaphase stage. The positions of centromeres are shown as relative distances from the spindle center d , as determined in the upper illustration. Zero and 1. The number of examined positions is shown in parentheses.
For rec12 mutant, only the attachment of homologous chromosomes to both poles is shown. D Dissociation of GFP-visualized sister centromeres cen2. Arrows indicate the SPB.
Arrowheads and barbed arrowheads show each of the homologous centromeres. 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. Recall that homologous chromosomes are not identical. They contain slight differences in their genetic information, causing each gamete to have a unique genetic makeup.
This randomness is the physical basis for the creation of the second form of genetic variation in offspring. Consider that the homologous chromosomes of a sexually reproducing organism are originally inherited as two separate sets, one from each parent. Using humans as an example, one set of 23 chromosomes is present in the egg donated by the mother.
The father provides the other set of 23 chromosomes in the sperm that fertilizes the egg. Every cell of the multicellular offspring has copies of the original two sets of homologous chromosomes. In prophase I of meiosis, the homologous chromosomes form the tetrads. In metaphase I, these pairs line up at the midway point between the two poles of the cell to form the metaphase plate.
Because there is an equal chance that a microtubule fiber will encounter a maternally or paternally inherited chromosome, the arrangement of the tetrads at the metaphase plate is random.
Any maternally inherited chromosome may face either pole. Any paternally inherited chromosome may also face either pole. The orientation of each tetrad is independent of the orientation of the other 22 tetrads. This event—the random or independent assortment of homologous chromosomes at the metaphase plate—is the second mechanism that introduces variation into the gametes or spores.
In each cell that undergoes meiosis, the arrangement of the tetrads is different. The number of variations is dependent on the number of chromosomes making up a set. There are two possibilities for orientation at the metaphase plate; the possible number of alignments therefore equals 2 n , where n is the number of chromosomes per set. Humans have 23 chromosome pairs, which results in over eight million 2 23 possible genetically-distinct gametes.
This number does not include the variability that was previously created in the sister chromatids by crossover. 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.
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