Condensins promote chromosome recoiling during early anaphase to complete sister chromatid separation

Abstract

Sister chromatid separation is initiated at anaphase onset by the activation of separase, which removes cohesins from chromosomes. However, it remains elusive how sister chromatid separation is completed along the entire chromosome length. Here we found that, during early anaphase in Saccharomyces cerevisiae, sister chromatids separate gradually from centromeres to telomeres, accompanied by regional chromosome stretching and subsequent recoiling. The stretching results from residual cohesion between sister chromatids, which prevents their immediate separation. This residual cohesion is at least partly dependent on cohesins that have escaped removal by separase at anaphase onset. Meanwhile, recoiling of a stretched chromosome region requires condensins and generates forces to remove residual cohesion. We provide evidence that condensins promote chromosome recoiling directly in vivo, which is distinct from their known function in resolving sister chromatids. Our work identifies residual sister chromatid cohesion during early anaphase and reveals condensins' roles in chromosome recoiling, which eliminates residual cohesion to complete sister chromatid separation.

Graphical abstract

Figure 1

Chromosome Arms Show Regional Stretching and Subsequent Recoiling during Their Segregation (A) Observation of three loci along chromosome XV during anaphase. Cells (T4189) with TetR-GFP and tetOs, integrated at three loci as indicated in (Ai), were arrested by α factor treatment and released into fresh medium. After 80 min, GFP images were collected every 4 s for 45 min. (Aii) Representative time-lapse images showing segregation of the three GFP-labeled loci during anaphase. Pink, yellow, and white circles indicate sister CENs (pulled toward opposite spindle poles), HIS, and TEL (on the sister chromatid that entered the bud), respectively. Time 0 is set arbitrarily. See Movie S1. Figure S1A shows images of other cells. (Aiii) Changes in the distances between the individual GFP-labeled loci. (Aiv) Schematic drawing of the segregating GFP-labeled loci. (B) Chromosome stretching between three different pairs of GFP-labeled loci was evaluated as in (A). (Bi) T4189, T6756, and T6876 cells carry each marked chromosome XV from left to right. (Bii) The maximum distances between the two GFP-labeled loci during their segregation in anaphase. Thick lines indicate mean values. (Biii) The maximum distances, averaged per 10 kb. Standard compaction was calculated, assuming that 10 kb spans 60–80 nm (Bressan et al., 2004; Bystricky et al., 2004). (C) Model of chromosome segregation during anaphase, accompanied by regional stretching and subsequent recoiling of a chromosome arm.

Figure 2

Sister Chromatid Cohesion Is Still Partially Present after Anaphase Onset and Contributes to Chromosome Stretching (A) Sister chromatids remain partially associated following the onset of anaphase. Cells (T7091) with TetR-GFP 3×CFP-LacI tetOs lacOs (integrated as indicated in [Ai], top) were arrested by α factor treatment and released into fresh medium. After 80 min, GFP and CFP images were collected every 30 s for 45 min. (Ai) Schematic drawing and (Aii) representative time-lapse images (time 0, start of image acquisition) showing segregation of CEN (tetOs; green), HIS (tetOs; green), and ADE (lacOs; red) loci. During the period (blue and orange rectangles) after anaphase onset (distance between sister CENs >3 μm) but before ADE CFP dots had begun segregating toward the poles (distance between sister ADEs <1.5 μm), it was scored whether the sister-HIS GFP dots remained associated (blue rectangles) or they showed separation at least for one time point (orange rectangles). (B) Residual cohesion during early anaphase partly depends on the cohesin complex and causes chromosome arm stretching. scc1-aid (T8455) and SCC1⁺ (T8487) cells with Pmet-CDC20 osTIR1 TetR-GFP 3×CFP-LacI tetOs lacOs (at CEN, ADE, and HIS as in [Ai]) were arrested with α factor treatment, released into fresh medium, and arrested at metaphase by Cdc20 depletion for 2.5 hr. Cells were released into anaphase by re-expression of Cdc20 (by transfer to methionine drop-out medium) concomitantly with addition of NAA. Subsequently, GFP and CFP images were collected every 30 s for 45 min. T8455 cells were also treated without NAA addition. In all three conditions in (Biii), the distance between sister CENs was enlarged (>3 μm) with similar timing (data not shown). (Bi) Schematic of auxin degron system. SCF: Skp1, Cullin, and F-box complex. E2: E2 ubiquitin ligase. aid: auxin-inducible degron. See Nishimura et al. (2009) for details. (Bii) Experimental procedure. (Biii) The separation of sister HIS GFP dots, scored as in (A). Figure S2D shows the amount of Scc1-aid protein during the time course. (Biv) The maximum distances between ADE and HIS during their segregation in anaphase of individual cells. Thick lines indicate mean values.

Figure 3

Chromosome Recoiling Is Inefficient or Defective during Anaphase in Condensin Mutants Condensin mutants (smc2-8; T3829 and ycg1-2; T3992) and wild-type control cells (T3790) with Pgal-CDC20 TetR-GFP tetOs (integrated at three loci as in Figure 1A) were treated with α factor at 25°C and then released into fresh medium at 35°C (restrictive temperature for smc2-8 and ycg1-2) and arrested at metaphase by Cdc20 depletion for 2.5 hr. Synchronous anaphase was then induced by re-expression of Cdc20 and GFP images were acquired every 4 s for 30 min, both at 35°C. (A) GFP signals were tracked as in Figure 1A. Graphs show the CEN-CEN (red) and CEN-HIS (blue) distances (time 0: CEN-CEN distance reached 3 μm). Time required for HIS segregation is defined as the period from time 0 until the CEN-HIS distance became <1.8 μm in the bud. Movies S2–S4 and Figure S3B also concern these cells. (B) The percentage of cells, in which HIS segregation (defined as in A) completed within the indicated time window. n = number of observed cells. (C) The segregation speed of the HIS locus toward the bud (rate of CEN-HIS shortening) was measured in T3790 (wild-type control) and T3829 (smc2-8) cells as indicated in (A). Thick lines indicate mean values.

Figure 4

Condensins Localize on Anaphase Chromosomes and Are Required during Anaphase for Their Recoiling and Segregation (A) Condensins localize on non-rDNA chromosomal arms during anaphase. Cells (T6971) with SMC4-3×GFP YCG1-3×GFP NET1-4×mCherry Ndc80-3×CFP were treated as in Figure 1. After 80 min, GFP, CFP, and mCherry images were acquired every 1 min for 45 min. Representative time-lapse images are shown (0 min: start of image acquisition). The cell shape is outlined in white at 0 min. Pink arrows indicate condensins (green) localizing between kinetochore clusters (white) during anaphase before rDNA (red) segregation (bidirectional arrow). (B) smc2-aid (T8636) and SMC2⁺ (T8429) cells with osTIR1 Pmet-CDC20 TetR-GFP tetOs (integrated at three loci as in Figure 1A) were treated with α factor, then released into fresh medium and arrested at metaphase by Cdc20 depletion for 2.5 hr. NAA was added concomitantly with Cdc20 re-expression (by transfer to methionine drop-out medium) and subsequently, GFP images were acquired every 4 s for 45 min. T8636 cells were also treated without NAA addition. In all three conditions (see Biii), the distance between sister CENs was enlarged (>3 μm) with similar timing (data not shown). (Bi) Schematic of experimental procedure. (Bii) Representative time-lapse images showing the behavior of the three GFP-labeled loci during anaphase. Pink, yellow, and white circles indicate sister CENs, HIS, and TEL, respectively, as in Figure 1Aii. Time 0: CEN-CEN distance reached 3 μm. Movies S5 and S6 concern the cells shown here. (Biii) The percentage of cells, in which HIS segregation (defined as in Figure 3A) completed within the indicated time window. n = number of observed cells. (Biv and Bv) The segregation speed of the HIS and TEL loci toward the bud (rate of CEN-HIS and CEN-TEL shortening), obtained by linear regression (as shown in Figures 3A and 5B), for cells that completed HIS or TEL segregation, respectively. Thick lines indicate mean values. Figure S4D shows the amount of Smc2-aid protein during the time course.

Figure 5

The Role of Condensins in Chromosome Segregation Is Not Limited to Resolution of Sister Chromatids (A) Condensin mutants show different behaviors of a chromosome arm locus, compared with a top2 mutant. smc2-8 (T3829), ycg1-2 (T3992), top2-4 (T3794), and top2-4 smc2-8 (T3936) cells with Pgal-CDC20 TetR-GFP tetOs (integrated at three loci as in Figure 1A) were treated and analyzed as in Figure 3. (Ai) Graphs show the CEN-HIS distances (time 0: CEN-CEN distance [not shown] reached 3 μm), for two representative top2-4 cells (#1 and 2) that completed HIS segregation with similar timing to the majority of smc2-8 cells (top); and for two representative top2-4 cells (#3 and 4) that did not complete HIS segregation, similarly to many ycg1-2 cells (middle). Finally, two top2-4 cells (#3 and 4) were compared with two representative top2-4 smc2-8 cells (bottom). The smc2-8 #1 and ycg1-2 #1 cells were also analyzed in Figure 3A. Figures S3B and S5A and Movies S2–S4 and S7 also concern the cells shown here. See Figure 3A (blue line) for the change in CEN-HIS distance in a representative “wild-type” cell. (Aii) The percentage of cells, in which HIS segregation completed (CEN-HIS distance became <1.8 μm without subsequently exceeding 3 μm) within the indicated time window. Compare with the results in “wild-type,” smc2-8, and ycg1-2 cells, shown in Figure 3B. n = number of observed cells. (B) The speed of telomere segregation is lower in condensin mutants. smc2-8 (T3829), ycg1-2 (T3992), and control wild-type cells (T3790; see their genotypes in A and Figure 3) were treated and analyzed as in Figure 3. (Bi) Changes in CEN-TEL distance in representative cells. Time 0 is set arbitrarily. (Bii) The speed of TEL segregation (shortening of CEN-TEL distance) toward the bud in individual cells. Thick lines indicate mean values.

Figure 6

Condensins Play Active Roles in Chromosome Recoiling Independently of Sister Chromatid Resolution/Separation (A) Making an unreplicated chromosome with two centromeres. YCG1⁺ (condensin wild-type, T5822) and ycg1-2 (T5823) cells with Pgal-CDC6 Pmet-CDC20 Pgal-eCEN3 (ectopic CEN3; integrated 120 kb to the left of the authentic CEN4) TetR-3×CFP GFP-LacI lacOs tetOs (integrated as shown in [Ai]) were arrested in metaphase by Cdc20 depletion and then released to anaphase (0 min in [Aii] FACS analyses) by re-expression of Cdc20 at 25°C. From 30 min before re-expression of Cdc20, Cdc6 expression was inhibited and eCEN3 was activated in glucose-containing medium. Cells were then arrested by α factor treatment, followed by release (90 min in [Aii]) into fresh medium at 35°C. After 80 min, GFP and CFP images were acquired every 6 s for 20 min. (B) An unreplicated dicentric chromosome shows condensin-dependent stretching and recoiling. (Bi) Representative time-lapse images (time 0, start of image acquisition) showing tetOs (CFP) in red and lacOs (GFP) in green. Orange brackets indicate examples of stretching and recoiling of the region between the CFP and GFP dots. (Bii) Changes in the distance between the two dots in the cells shown in (Bi). (Biii) Frequency of large changes (≥0.9 μm within 18 s) in the distance between the two dots in individual cells. Thick lines indicate mean values. (C) Power spectra analyses for the oscillation of the CFP and GFP dots. (Ci) Using discrete Fourier transforms, power in the oscillation (red) was plotted as function of frequency of oscillation. Error bars (gray) show 90% confidence of the power. The power was fitted by curves, based on a motion of power-law (green), diffusion (purple), or damped elastic spring (cyan). See more detail in Supplemental Note for Power Spectrum Analyses. (Cii) Total power (variance) in individual cells. Thick lines indicate mean values.

Figure 7

Condensin-Dependent Chromosome Recoiling Facilitates Elimination of Residual Sister Chromatid Cohesion (A) A larger amount of cohesin remains on chromosomes during anaphase when chromosome recoiling is defective. YCG1⁺ (T7802) and ycg1-2 (T7803) cells with Pgal-CDC20 SCC1-18×myc TetR-GFP tetOs (at CEN and TEL loci as in Figure 1A) were arrested at metaphase by Cdc20 depletion and subsequently released to anaphase synchronously by re-expression of Cdc20, as in Figure 3. During metaphase arrest and also 10 min after Cdc20 re-expression, chromosomes were fixed and immobilized on a slide glass immediately after cell lysis. Chromosomes were stained with DAPI and Scc1 was immunostained using an anti-myc antibody. (Ai) Representative cells. (Aii) The distance between sister CEN dots and the percentage of cells, in which TEL-dot segregation was completed (toward two distinct nuclear masses). (Aiii) The amount of Scc1, bound on chromosomes, was quantified and compared between the two strains. Bars and errors show means and SEMs, respectively. n.s. = not significantly different. (B) Reduction of residual cohesion restores chromosome segregation in condensin-defective cells. smc2-aid (T8636) and smc2-aid scc1-aid (T8595) cells with osTIR1 Pmet-CDC20 TetR-GFP tetOs (integrated at three loci as in Figure 1A) were induced to synchronous anaphase by depletion of Cdc20 followed by its re-expression, as in Figure 4B. NAA was added concomitantly with Cdc20 re-expression and after 15 min GFP images were acquired every 4 s for 45 min. The graph shows the time (after the CEN-CEN distance became >3 μm) when TEL segregation occurred to the bud. As a control, metaphase was extended for T8595 cells without re-expression of Cdc20 but with addition of NAA in the same timing; CEN-CEN distance did not exceed 3 μm during image acquisition (data not shown). (C) Summary for residual sister chromatid cohesion and its elimination by condensin-dependent chromosome recoiling. Separase cleaves the majority of cohesin rings at the onset of anaphase. However, due to a small amount of residual cohesins, weak sister chromatid cohesion is still present at some loci along chromosome arms, which transiently opposes sister chromatid separation and causes regional chromosome stretching. Stretched chromosome regions are recoiled by the action of condensins, leading to removal of residual cohesins, either by their cleavage facilitated by separase (Ci) or by their physical breakage/removal (Cii and Ciii) (see Discussion). Regional chromosome stretching/recoiling advances from para-centromere regions to telomeres, resulting in sister chromatid separation along the entire chromosome arms.