Chiasmata promote monopolar attachment of sister chromatids and their co-segregation toward the proper pole during meiosis I

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. Chiasmata are essential for the attachment of the homologous chromosomes to opposite spindle poles (bipolar attachment) and their subsequent segregation to the opposite poles during meiosis I. However, the overall function of chiasmata during meiosis is not fully understood. Here, we show that chiasmata also play a crucial role in the attachment of sister chromatids to the same spindle pole and in their co-segregation during meiosis I in fission yeast. Analysis of cells lacking chiasmata and the cohesin protector Sgo1 showed that loss of chiasmata causes frequent bipolar attachment of sister chromatids during anaphase. Furthermore, high time-resolution analysis of centromere dynamics in various types of chiasmate and achiasmate cells, including those lacking the DNA replication checkpoint factor Mrc1 or the meiotic centromere protein Moa1, showed the following three outcomes: (i) during the pre-anaphase stage, the bipolar attachment of sister chromatids occurs irrespective of chiasma formation; (ii) the chiasma contributes to the elimination of the pre-anaphase bipolar attachment; and (iii) when the bipolar attachment remains during anaphase, the chiasmata generate a bias toward the proper pole during poleward chromosome pulling that results in appropriate chromosome segregation. Based on these results, we propose that chiasmata play a pivotal role in the selection of proper attachments and provide a backup mechanism that promotes correct chromosome segregation when improper attachments remain during anaphase I.

Figure 1. Spindle attachment of chromosomes and their segregation during mitosis and meiosis I.

For simplicity, only a single kinetochore-interacting microtubule is shown for each kinetochore, and other microtubules are not shown.

Figure 2. The effect of loss of chiasmata on chromosome segregation.

(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. +: no sgo1 mutation; cen1: the lys1 locus; cen2: the D107 locus ; rec+: no rec12 or rec14 mutation. 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. +: sgo1⁺ cells; sgo1Δ: sgo1 mutant cells. The lower illustrations in (A), (B), and (D) show how GFP was used to mark chromosomes (left) and the segregation patterns of the GFP signals after meiosis I (right). 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. *p<0.05; ** p<0.005; *** p<5×10⁻⁵.

Figure 3. A role for the SAC factor, Mad2, in chromosome segregation during meiosis I.

(A) Effects of Mad2 depletion on sister chromatid segregation during meiosis I in rec12 achiasmate cells. +: no mad2 mutation. (B) Effects of Mad2 depletion on sister chromatid segregation at meiosis I in haploid cells. Sister chromatid segregation was analyzed using GFP-visualized cen2. (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). Numbers indicate the time in minutes from the beginning of spindle formation. Bar: 2 µm. 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. Error bars indicate standard deviation. Asterisks show statistically significant differences (p<0.05).

Figure 4. Pre-anaphase centromere dynamics during meiosis I in wild-type and rec12 mutant cells.

(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. Vertical bar: 2 µm. Graphs show changes in the distance between the SPB and each centromere (red, SPB#1-cen#1, and blue, SPB#1-cen#2) and between the two SPBs (black, SPB#1-SPB#2). (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 1.0 correspond to positions of the spindle center and the SPB, respectively. The number of examined positions is shown in parentheses. (C) Spindle attachment of chromosomes during meiosis I in wild-type (Wt) and rec12 mutants (rec12Δ) and expected observation frequencies of centromeres at distinct positions in the spindle. 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. Bar: 1 µm. (E) Average dissociation frequencies of sister centromeres in wild-type and rec12 mutant cells. The number of centromeres examined is shown in parentheses. (F) Dissociation of sister centromeres visualized by GFP-tagged Cnp1 during the pre-anaphase stage. 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. Bar: 1 µm. 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. Error bars indicate standard deviation.

Figure 5. Sister chromatid segregation in 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. +: no rec12 or mad2 mutation. (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. Error bars indicate standard deviation. Asterisks show statistically significant differences and their associated p values. * p<0.05; ** p<0.005; *** p<0.0005; **** p<5×10⁻⁶.

Figure 6. Pre-anaphase centromere dynamics during meiosis I in mrc1 and moa1 mutants.

(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. Horizontal bar: 50 s. Vertical bar: 2 µm. (B) Average centromere dissociation frequencies in mrc1, moa1, and rec8 mutant cells. The number of centromeres examined is shown in parentheses. +: no rec12 mutation. ND: not determined. Asterisks indicate dissociation frequencies that are statistically different from the frequency of wild type. * p<0.005; ** p<0.01. (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. The number of examined positions is shown in parentheses. (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 asterisk indicates a distance statistically different from that of wild type (p<5×10⁻¹²⁵). 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.

Figure 7. Centromere dynamics during anaphase I in mrc1 and moa1 mutants.

Arrows and arrowheads show each of the homologous centromeres (cen2), respectively, and the two arrowheads or arrows indicate dissociated sister centromeres. Horizontal bar: 50 s. Vertical bar: 2 µm.

Figure 8. Two major roles of chiasmata during meiosis I.

(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.