Spindle architecture constrains karyotype evolution

Abstract

The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome numbers vary dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.

Fig. 1. Chromosome fusions induce spindle defects from 1n = 4.

a, Chromosome lengths of wild-type (16 chr) and 3 chr S. cerevisiae. Vertical lines indicate positions of centromeres. b, Maximum growth rates of fused-chromosome strains on synthetic complete medium with 2% dextrose (SCD). Boxes show the means and s.d. Means were compared with the wild type using a two-tailed Student’s t-test (from left to right, n = 8, 8, 8, 8, 8, 7, 8 and 8 independent population measurements). c, Montage of SPB (Spc42-mCherry) dynamics during mitosis for 16- and 3-chromosome strains. Scale bar, 2 µm; intervals are 3 min. The time point with maximal SPB separation during anaphase was set to zero. Open arrows indicate SPB doubling. d, Distance between SPBs over time. For normalization, the time point with maximal SPB separation during anaphase was set to zero. The vertical line represents the inflection point (~start of anaphase). e, The time from SPB doubling to max. anaphase separation. Boxes represent the mean and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 18, 18, 20, 39 and 34 independent single-cell measurements). chr, chromosome. Source numerical data are available in source data. Source data

Fig. 2. Defects are overcome by diploidization during experimental evolution.

a, Schematic overview of the evolution experiments. Replicate populations of strains with either 16, 8 or 3 chromosomes were inoculated in 96-well plates filled with 100 µl SCD and 1:100 of each culture was transferred daily for a total of ~150 generations. b, Maximum growth rate on SCD over the course of evolution separated by genotype. Curves are smoothed and represent the average trend of eight replicate evolving populations. Ribbons represent 95% confidence intervals. c, Mutations observed in selected evolved strains. The number of mutations per sequenced strain is shown on the left, the proportions of homozygous (Hom.) and heterozygous (Het.) mutations are shown in green and the pink charts show the proportion of nonsense mutations and frameshifts (STOP), nonsynonymous (Nonsyn.) mutations, synonymous (Syn.) mutations and intergenic mutations. d, Representative pulsed-field gel electrophoresis (PFGE) gel showing the karyotype of the three ancestral genotypes and 11 evolved three-chromosome strains. The remaining four gels can be found in Extended Data Fig. 2. e, The proportion of ploidies observed in evolved strains separated by genotype. For evolved 16- and 8-chromosome strains, clones from 16 populations were checked for ploidy. For evolved three-chromosome strains, all 56 populations were checked for ploidy. f, To make diploids, the mating type was switched using a plasmid with inducible HO endonuclease, and cells were allowed to mate to form diploids (left), maximum growth rates of haploid and diploid fused-chromosome strains (middle) and epistasis between chromosome number and ploidy (right). Boxes represent the means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 16, 16, 15, 13, 15 and 13; independent population measurements). Source numerical data and unprocessed gels are available in source data. Source data

Fig. 3. Five centromeres are sufficient to overcome the mitotic defect.

a, Maximum growth rates of fused-chromosome strains with and without additional centromere (red circle), supplied on either a plasmid or a small artificial chromosome. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 16, 16, 16, 11, 15, 16, 15, 16 and 12 independent population measurements). b, Maximum growth rates of haploid and diploid fused-chromosome strains. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 11, 16, 26, 12, 13, 15, 23 and 12 independent population measurements). c, Distance between SPBs over time for the four-chromosome strain with and without centromeric plasmid. For normalization, the time point with maximal SPB separation during anaphase was set to zero. d, The time from SPB doubling to max. anaphase separation for the four-chromosome strain with and without centromeric plasmid. Boxes represent the means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 20, 39 and 18 independent single-cell measurements). e, The time from SPB doubling to max. anaphase separation for diploid strains. Boxes represent the means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 18, 18, 20 and 17 independent single-cell measurements). WT, wild type. Source numerical data are available in source data. Source data

Fig. 4. Decreasing the net outward force in the mitotic spindle alleviates the defect.

a, Simplified schematic of inward (green arrows) and outward (pink arrows) forces in a metaphase spindle. b, Distance between SPBs at the end of metaphase for different fusion strains. c, Maximum growth rates of fused-chromosome strains with and without benomyl. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 16, 16, 16 and 15 independent population measurements). d, The time from SPB doubling to max. anaphase separation. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 35, 34 and 18 independent single-cell measurements). e, Maximum growth rates of fused-chromosome strains with and without KIP1 deletion. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 15, 14, 13 and 15 independent population measurements). f, Summary of epistatic effects of different perturbations. Diploidization, adding a centromeric plasmid or adding an artificial chromosome increase the inward force and adding benomyl or deleting KIP1 decrease the outward force. Boxes represent means and s.d. g, Distance between SPBs at the end of metaphase for different fusion strains and the effects of increasing inward force (+pCEN) or decreasing outward force (+benomyl). Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 20, 39, 18, 34 and 18 independent single-cell measurements). Source numerical data are available in source data. Source data

Fig. 5. The force imbalance causes kinetochore declustering and triggers the SAC.

a, Maximum growth rates of fused-chromosome strains with and without MAD2 deletion. Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 14, 13, 15 and 16 independent population measurements). b, The time from SPB doubling to max. anaphase separation. Boxes represent the means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 17, 14, 15 and 13 independent single-cell measurements). c, Maximum growth rates of fused-chromosome strains with and without MAD2 deletion + benomyl treatment (55 µM). Boxes represent means and s.d. Means were compared using a two-tailed Student’s t-test (from left to right, n = 15, 15, 14 and 13 independent population measurements). d, Western blot analysis of Pds1 levels after G1 release for 16- and 3-chromosome strains. Cells were collected at the indicated time points and alpha-factor pheromone was added again 45 min after release to prevent the cells from entering a second cell cycle. Actin levels were used as a loading control. Pds1-normalized values are shown in the bar plot at the bottom. An independent repeat of this experiment with analysis of the second cell cycle can be found in Extended Data Fig. 5f. e, Proportion of cells with a bi-lobed kinetochore signal (bi-modal Ndc80 signal along the spindle pole to spindle pole axis) in both metaphase and anaphase. Proportions were compared using a two-proportions z-test (from left to right, n = 26, 56, 100, 24, 41 and 31 independent single-cell measurements). f, Montage of kinetochore (Ndc80-mNG) and SPB (Spc42-mCherry) dynamics during metaphase for 16- and 3-chromosome strains. Scale bar, 2 µm; intervals are 30 s. g, Montage of kinetochore (Ndc80-mNG) and SPB (Spc42-mCherry) dynamics during metaphase in three-chromosome strains with benomyl (55 µM). Scale bar, 2 µm; intervals are 1 min. Source numerical data and unprocessed blots are available in source data. Source data

Extended Data Fig. 1. Growth and mitotic defects in fused-chromosome strains.

(a) Maximum growth rates of fused-chromosome strains on synthetic complete medium with 2% dextrose (SCD). Boxes show the means and standard deviation. Means were compared using a two-tailed Student’s t-test (from left to right, n = 8, 7, 7, 8, 8, 8, 7, 5, 7, 5; independent population measurements). (b) Distance between SPBs over time for different fusion strains. For normalization, the time point with maximal SPB separation during anaphase was set to zero. (c) Expanded cells with spindle defects. Cells were labelled with pan protein label NHS ester and for tubulin. Scale bar = 10 µm, expansion factor = 4.18. (d) Montage of spindle dynamics over time (CloverGFP-tub1). Scale bar = 5 µm, intervals are 1 min. Closed arrows point to an example of increased spindle curvature, open arrows to an example of the whole spindle moving into the daughter cell. (e) Spindle curvature (%), calculated as the total spindle length relative to the distance between spindle pole bodies (SPBs). Data are represented as violin plots with boxes representing the interquartile range, the lines representing the median and the whiskers indicating the minimum and maximum values. Black dots represent outliers. n = 50 for each genotype; independent single-cell measurements. Distributions were compared using Kolmogorov-Smirnov tests. (f) Montage of nuclear envelope (Hmg1-mCherry) dynamics over time. Scale bar = 5 µm, intervals are 1 min. Source numerical data are available in source data. Source data

Extended Data Fig. 2. PFGE of evolved strains shows no chromosome fission.

PFGE gels showing the karyotype of the evolved 3-chromosome populations. Red stars indicate populations with cross-contamination of a wild-type strain. Chromosome numbers of clones isolated from these populations were double-checked before ploidy determination and sequencing. Unprocessed gels are available in source data. Source data

Extended Data Fig. 3. Distance between SPBs over time in diploids.

For normalization, the time point with maximal SPB separation during anaphase was set to zero. Source numerical data are available in source data. Source data

Extended Data Fig. 4. Distance between SPBs after changing the net outward force in the metaphase spindle.

(a) Distance between SPBs at the end of metaphase for different diploid fusion strains. Boxes represent means and standard deviation (from left to right, n = 18, 18, 20, 17; independent single-cell measurements). (b) Distance between SPBs over time in benomyl-treated cells. For normalization, the time point with maximal SPB separation during anaphase was set to zero (top: n = 20, bottom: n = 18). Source numerical data are available in source data. Source data

Extended Data Fig. 5. SAC activation in fused-chromosome strains.

(a) Epistatic effect of MAD2 deletion. Boxes represent means and standard deviation. (b) Maximum growth rates of fused-chromosome strains with and without RAD9 deletion. Boxes represent means and standard deviations. Means were compared using a two-tailed Student’s t-test (from left to right, n = 16, 16, 16, 16; independent population measurements). (c) Maximum growth rates of fused-chromosome strains with and without 10 mM hydroxyurea (HU). Boxes represent means and standard deviations. Means were compared using a two-tailed Student’s t-test (from left to right, n = 13, 16, 16, 15; independent population measurements). (d) Maximum growth rates of fused-chromosome strains with and without BUB2 deletion. Boxes represent means and standard deviations. Means were compared using a two-tailed Student’s t-test (from left to right, n = 19, 21, 21, 21; independent population measurements). (e) Distance between SPBs over time in MAD2∆ cells. For normalization, the time point with maximal SPB separation during anaphase was set to zero. (f) Western blot analysis of Pds1 levels after G1 release for 16- and 3-chromosome strains, focused on the second cell cycle after release. Cells were collected at the indicated time points. Ponceau S staining was used as a loading control. Pds1-normalized values are shown in the bar plot at the bottom. An independent repeat of this experiment with analysis of the first cell cycle can be found in Fig. 5d. Source numerical data and unprocessed blots are available in source data. Source data