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A Midzone-Based Ruler Adjusts Chromosome Compaction to Anaphase Spindle Length

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Science  22 Apr 2011:
Vol. 332, Issue 6028, pp. 465-468
DOI: 10.1126/science.1201578

Abstract

Partitioning of chromatids during mitosis requires that chromosome compaction and spindle length scale appropriately with each other. However, it is not clear whether chromosome condensation and spindle elongation are linked. Here, we find that yeast cells could cope with a 45% increase in the length of their longest chromosome arm by increasing its condensation. The spindle midzone, aurora/Ipl1 activity, and Ser10 of histone H3 mediated this response. Thus, the anaphase spindle may function as a ruler to adapt the condensation of chromatids, promoting their segregation regardless of chromosome or spindle length.

During animal cell division, chromosome partitioning requires mitotic chromosomes to be compact enough to allow their segregation; conversely, the spindle must elongate enough to segregate even the longest chromosome (1, 2). To address how cells cope with a highly oversized chromosome, we generated a very long chromosome arm (where “chromosome length” refers to nucleotide number). The two longest chromosomes (IV and XII) of budding yeast were fused by homologous recombination in vivo to form the long compound chromosome LC(XII:IV) (Fig. 1A) (3). To prevent dicentric chromosome formation, the centromere CEN4 was inactivated in galactose-containing medium by the GAL1-10 promoter (4). GAL1:CEN4 cells grew poorly in galactose, due to chromosome IV instability (Fig. 1B). In contrast, LC(XII:IV)GAL:CEN4 cells grew well in galactose, consistent with segregation of chromosome IV with CEN12, whereas they grew poorly in glucose, owing to dicentric chromosome formation (Fig. 1B). We next generated the true monocentric compound chromosomes LC(XII:IV)cen4∆ and LC(XII:IV)cen12∆ by deleting CEN4 and CEN12, respectively (fig. S1). Whole genome sequencing showed that gene order and intergenic distances were conserved between wild-type and LC(XII:IV) strains and identified no genomic rearrangements other than the fusion of chromosomes IV and XII (fig. S2). Quantitative polymerase chain reaction (qPCR) analysis indicated that the number of ribosomal DNA (rDNA) repeats was reduced by 6 to 25% in LC(XII:IV)cen4∆ strains relative to wild-type chromosome XII (87 to 110 repeats versus 110 to 120) (fig. S3). This established that even after rDNA array shortening the long arm of LC(XII:IV) was 45% longer than the longest wild-type arm (3.2 versus 2.2 Mb). Thus, yeast cells tolerate remarkably well a large increase in the length of their longest chromosome arm, although they have probably not experienced it naturally over the past 100 million years (3).

Fig. 1

Segregation of a long compound chromosome. (A) Chromosome fusion through recombination of a bridging fragment [see (3) for details]. Circles indicate centromeres (black), TRP1 (red), and LYS4 (green) loci; numbers indicate estimated distances (Mb) to the active centromere. Chromosomes are not to scale. (B) Serial dilutions of log-phase cultures at 30°C for 48 hours (glucose) or 72 hours (galactose). Galactose inactivates the conditional centromere (pGAL). (C and D) Analysis of SPBs (green, Spc42-GFP, asterisks), LYS4 (green), and TRP1 (red) trajectories in wild-type and LC cells. Bud-directed chromatids are pulled away from their sisters in the mother cell, which move comparatively less. Arrowheads and arrows indicate segregation of TRP1 and LYS4, respectively. Time 0, anaphase onset. Scale bars, 1 μm. (E) Spindle elongation dynamics (distance between SPBs) in wild-type and LC(XII:IV)cen4∆ cells (N = 20). The arrow marks spindle breakdown. (F) Time of segregation (sister spots are separated by >2 μm) relative to anaphase onset (left), or corresponding spindle length (right), for loci on the indicated chromosome, plotted against the distance to the centromere. Nonlinear fit shows an asymptotic relationship (N > 25). In (E) and (F), data points are mean ±SEM.

We next examined how cells segregate LC(XII:IV) chromosomes. Anaphase spindle elongation, visualized by fusing the spindle pole body (SPB) component Spc42 to green fluorescent protein (Spc42-GFP), progressed with identical kinetics in wild-type and LC(XII:IV)cen4∆ cells, to reach identical lengths (Fig. 1, C to E). Thus, to segregate this long chromosome, cells did not need to elongate the spindle more or prolong anaphase.

Chromosome IV was next visualized using Tet and Lac operator arrays integrated at the TRP1 and LYS4 loci (Fig. 1A), and TetR-mRFP and LacI-GFP reporters (5) (Fig. 1, C and D, and fig. S4). Segregation of the TRP1 and LYS4 loci in LC(XII:IV)cen4∆ cells occurred in inverse order than in wild type, consistent with inversion of chromosome IV sequences relative to the active centromere (Fig. 1F). Furthermore, segregation of TRP1 and LYS4 in the compound chromosome occurred after the bulk of spindle elongation, suggesting that it depended on an increase in chromosome compaction, consistent with (6) (Fig. 1F). Accordingly, LC(XII:IV) cells carrying the conditional condensin mutation smc2-8 showed defects in LC(XII:IV) segregation and reduced viability at semipermissive temperature, compared with smc2 mutants of normal karyotype (fig. S5). To investigate whether LC(XII:IV) condensation was indeed increased relative to chromosome IV, we used the distance between the TRP1 and LYS4 loci, which shortens in a condensin-dependent manner during mitosis (5), as a compaction reporter. To ensure that inactivation of the nearby centromere would not affect the analysis, the TRP1-LYS4 distance was measured on the LC(XII:IV)cen12∆ chromosome, keeping CEN4 intact (Fig. 2A). In wild-type cells, the TRP1-LYS4 distance decreased after segregation (Fig. 2, B and C) to about 75% of its metaphase value, indicating that chromosome IV compaction still increased in late anaphase. Anaphase compaction was systematically highest for the bud-directed chromatid (Fig. 2C) and further increased in the LC(XII:IV)cen12∆ cells (Fig. 2C). Thus, chromosome lengthening triggered hypercompaction of the TRP1-LYS4 region in late anaphase. Hypercondensation required condensin function (fig. S5) and spread over CEN-proximal (Fig. 2C) and CEN-distal regions (fig. S6) of the compound arm. We termed this response “adaptive hypercondensation.”

Fig. 2

Anaphase condensation is increased in the long compound chromosome. (A) Representative metaphase (left) and anaphase (right) cells with labeled SPBs (large green dot), TRP1 (red), and LYS4 (green) loci. The positions of the fluorescent marks on the chromosomes are indicated (top) as in Fig. 1. (B) Distance between the TRP1 and LYS4 spots [∆x in (A)] measured in time series in mother cells. (C) TRP1-LYS4 distances during metaphase (10 min before anaphase onset) and anaphase [shaded region in (B)] in indicated compartments (N > 25). In these and the following graphs, boxes include 50% of data points, whiskers 95%. Median (lines) and mean (crosses) are shown. Asterisks indicate P < 0.02 (*) or P < 0.001 (**). (D) TRP1-LYS4 distances [as in (C)] in depicted diploid cells (N > 20).

To determine whether adaptive hypercondensation affected only LC(XII:IV) or all chromosomes, we analyzed condensation of the TRP1-LYS4 region in diploids where one copy each of chromosomes IV and XII were fused (Fig. 2D). Whereas the compaction of the TRP1-LYS4 reporter increased during anaphase when it was placed on LC(XII:IV)cen12∆, it did not when placed on the nonfused copy of chromosome IV (Fig. 2D). Thus, cells appear to have a “chromosome ruler” to assess the length of individual chromosomes and adapt their condensation.

The chromosome passenger complex (CPC), comprising the aurora-B kinase and INCENP (Ipl1 and Sli15 in budding yeast), localizes to the spindle midzone (7) and regulates chromosome condensation during anaphase (5, 8, 9), suggesting that it might promote condensation in the vicinity of the midzone and that the spindle itself might be the ruler. Thus, we asked whether Ipl1 at the spindle midzone mediated the adaptive response. We characterized chromatin compaction in temperature-sensitive ipl1-321 mutants shifted to 35°C at anaphase onset. This treatment did not perturb spindle dynamics (7) (fig. S7) and only mildly affected kinetochore biorientation (10) (TRP1 sister-dots on chromosome IV segregated correctly in 100% of wild-type cells and 93% of ipl1-321 cells; N > 100). However, Ipl1 inactivation affected TRP1-LYS4 compaction, particularly in LC(XII:IV)cen12∆ and the bud-directed chromatids (Fig. 3A and fig. S8). Yet, Ipl1 inactivation did not abolish anaphase condensation in general (fig. S8). Thus, Ipl1 contributed little to the bulk of anaphase condensation but was required for hypercondensation of chromosome arms, proportionally to their length. Mutation of Ser10 to alanine in the two histone H3 genes HHT1 and HHT2 phenocopied Ipl1 inactivation, reducing condensation of LC(XII:IV)cen12∆ more than of chromosome IV, specifically in anaphase, and most strongly in the bud (Fig. 3B and fig. S10). Although we cannot rule out general effects on gene expression, these data support the idea that aurora-dependent phosphorylation of histone H3 at serine 10, which is thought to regulate chromatin structure (11), may mediate at least part of adaptative hypercondensation.

Fig. 3

Hypercondensation of the long chromosome requires Ipl1 activity and S10 histone H3 phosphorylation. Increase in TRP1-LYS4 distances, expressed as percentage relative to wild-type, in ipl1-321 cells at 35°C (A) and in cells expressing S10A alleles of the histone H3 HHT1 and HHT2 genes at 30°C (B). Error bars are SEM, calculated from data on figs. S8 and S10 by error propagation. *, P < 0.02; **, P < 0.001, relative to wild type.

Next, we asked whether adaptive hypercondensation required Ipl1 localization to the spindle midzone. The midzone protein Slk19 mediates timely activation of the Cdc14 phosphatase (12), CPC and separase spindle recruitment (13, 14), and midzone focusing (15). Loss of Slk19 caused a decrease in anaphase compaction of TRP1-LYS4 on chromosome IV (Fig. 4A), the second-longest yeast chromosome, and delayed LYS4 segregation (P < 0.02) (Fig. 4B). Proper Ipl1 localization is recovered in slk19∆ and cdc14 mutant cells when replacing Sli15 with its constitutively dephosphorylated form, Sli15-6A (13). Expression of SLI15-6A in slk19∆ cells restored chromosome compaction and segregation to near wild-type levels. Thus, Ipl1 must be on the midzone to adapt condensation of endogenous long chromosomes (Fig. 4, A and B). Because SLI15-6A did not rescue the localization of separase in slk19∆ cells (fig. S9), Ipl1 regulated condensation independently of separase localization.

Fig. 4

Ipl1 targeting to the spindle midzone is required for segregation of long chromosome arms. (A) TRP1-LYS4 distance in chromosome IV throughout anaphase, in cells of indicated genotype (mean ± SEM). (B) Segregation time of TRP1 and LYS4 in cells carrying native chromosome IV. (C) Representative image series showing segregation of the TRP1 locus, located at the distal tip of the long arm of LC(XII:IV)cen4∆. Arrowheads mark TRP1 before and after segregation. (D) Segregation time of TRP1 in LC(XII:IV)cen4∆ cells of the indicated genotype. (E) Percentage of cells in which the spindle breaks before chromosome segregation. All cells are diploid strains carrying one chromosome with labeled TRP1 and LYS4 loci (wild type, wt) and either homozygous for the SLK19 deletion (slk19) or slk19 homozygous mutants carrying one copy of SLI15 and one copy of the dominant SLI156A allele (slk19 SLI156A). Time-lapse series were acquired at 30°C. (N > 18 for Chr IV; N > 30 for LC).

To examine the effects of midzone-bound Ipl1 on the segregation of long chromosomes, we visualized the distal region of LC(XII:IV)cen4∆, marked by the TRP1 locus (2.8 Mb from CEN12), in slk19∆ mutants. Separation of the distal TRP1 locus, but not of a centromere-proximal one, was delayed in slk19∆ mutants (P < 0.001) (Fig. 4, B to D). As a consequence of this delay, spindle breakdown preceded TRP1 segregation in 27% of LC(XII:IV)cen4∆ slk19∆ mutant cells (Fig. 4, C and E). Expression of SLI15-6A largely suppressed these defects (Fig. 4E). Thus, Slk19 affected segregation mainly through targeting of Ipl1/Aurora-B to the spindle midzone, which was especially important for the segregation of long chromosomes.

Together, our results indicate that yeast cells adjust the compaction of chromosomes to secure their segregation by the spindle. One key component of the underlying “ruler” may be the anaphase spindle, acting through the kinase aurora B at the midzone. Because long chromosome arms are exposed longer to the midzone than short ones, this model (fig. S11) accounts for their increased compaction and explains why compaction is also greater in the daughter cell. This simple model could also explain how small cells, with short spindles, still segregate their chromosomes at mitosis. Indeed, small cells such as whi3∆ mutants (16) hypercondensed their native chromosome IV (fig. S12), indicating that natural chromosomes adapt their compaction to anaphase spindle length.

Large variations in cell size and spindle length are common within species, and hyperlong chromosomes are well tolerated, at least in Drosophila (17). Similarly, chromosome rearrangements can increase chromosome size without diminishing cellular proliferation during cancer (18) or during size variations of rDNA loci (19). Perhaps spindle length and the level of chromosome condensation are not predetermined but are mutually coordinated through feedback regulatory loops. The mechanism described here is likely to be only one of such coupling systems. These probably play important roles not only during cell size changes but also in allowing chromosome rearrangements during speciation (20) and the survival of chromosomally unstable cancers (21).

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1201578/DC1

Materials and Methods

SOM Text

Figs. S1 to S12

Table S1

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank P. Meraldi, Z. Shcheprova, C. Weirich, and S. Buvelot for helpful comments and critical reading of the manuscript; D. Clarke (University of Minnesota) for reagents; C. Iannone and R. Tejedor for help with qPCR; F. Campelo for help with statistical analysis; the ETH Light Microscopy Center; the CRG Advanced Light Microscopy Unit; and the CRG Ultrasequencing Unit. This project was supported by grants from La Caixa to G.N., the Spanish Ministry of Science to T.G. (BFU09-09168) and M.M. (BFU09-08213), and the Swiss National Science Foundation to Y.B.

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