The kinetochore ensures accurate chromosome segregation by attaching chromosomes to the microtubules of the spindle. The kinetochore is built on centromeric DNA and in mitosis, the two kinetochores of replicated sister chromatids attach to microtubules from opposite spindle poles1
. The opposing forces on the two kinetochores are resisted by cohesin molecules that encircle the two sister chromatids2
, creating tension at the kinetochores and stabilizing their binding to microtubules. If both kinetochores attach to the same pole, they sense the reduced tension and release their microtubules, activating the spindle checkpoint, delaying anaphase, and allowing another attempt at proper attachment. At the onset of anaphase, cohesin is cleaved and sister chromatids separate from one another, allowing their kinetochores to travel to the spindle poles by following the ends of depolymerizing microtubules1
The budding yeast centromere is 125 base pairs long, assembles one microtubule-binding site, and recruits more than 80 proteins. Many of these kinetochore proteins are conserved among eukaryotes3
and assemble into biochemically and genetically defined complexes. The highly conserved KMN network includes the Mtw1 and Ndc80 complexes, but the DNA-binding CBF3 complex and microtubule-binding Dam1-DASH complex are less conserved1
To understand how the kinetochore assembles, we asked if we could build a synthetic kinetochore in budding yeast. We recruited a lactose repressor (LacI)-kinetochore protein fusion to a plasmid that lacked a centromere but carried multiple tandem repeats of the lactose operator (LacO) () and monitored the segregation of this plasmid. Without a centromere, plasmids prefer to segregate to the mother cell at division and are lost at a high frequency4
; forming a synthetic kinetochore should dramatically reduce the rate at which the test plasmid is lost5
Assay for a synthetic kinetochore
Our initial assay measured the fraction of cells that contain the plasmid after growth in medium that does not select for the plasmid. Under these conditions, only 20% of cells contain a centromere-less plasmid, compared to 80% when the plasmid carries a centromere (). We screened 17 kinetochore protein-LacI fusions (Supplementary Table 1
). Only one, the fusion between the C-terminus of Ask1 and the N-terminus of LacI, enhanced the plasmid’s segregation: 65% of the cells carried the LacO-containing plasmid. We got similar results with plasmids containing 8 or 256 copies of LacO (Supplementary Table 2
Ask1 is an essential protein, which is a component of the 10-member Dam1-DASH complex1, 3
, whose association with the kinetochore depends on microtubules6–8
. The N-terminal half of the protein enhances plasmid segregation and complements a deletion of ASK1
(Supplemental Figure 1
) and localizes to spindle microtubules indistinguishably from full-length Ask1. This correlation suggests that Ask1-LacI recruits the microtubule-binding activity of the Dam1-DASH complex to the LacO array. Fusions of other members of the Dam1-DASH complex to LacI did not enhance plasmid segregation (Supplementary Table 1, 2
) even though several could replace the endogenous, essential gene. The observation by Kiermaier et al.
that a different fusion to Dam1 could rescue plasmid segregation suggests that some of our fusions are only partially functional9
The Ask1-LacI fusion nucleates a “synthetic kinetochore” on the LacO array that can replace the natural kinetochore and direct chromosome segregation. Chromosomes with two centromeres (dicentrics) are mitotically unstable10
, prompting us to ask if chromosomes carrying a natural and a synthetic kinetochore were lost frequently. In diploid cells, the wild type version of chromosome III is lost at a rate of 1 to 2 × 10−6
/cell/generation, but adding the synthetic kinetochore to this chromosome increased the loss rate 275 fold to 5.5 × 10−4
showing that the synthetic kinetochore can interfere with a natural kinetochore’s ability to direct chromosome segregation ().
The synthetic kinetochore can replace a natural kinetochore
We made the natural centromere repressible by placing the GAL1
promoter upstream of the centromere; transcription from a strong promoter towards the centromere inactivates the kinetochore11
. Adding galactose activates the GAL1
promoter, disrupting the natural kinetochore, which is fully functional in glucose-grown cells ().
We made haploid cells with the synthetic kinetochore on chromosome III, 23kb from the galactose-repressed CEN3. In the absence of Ask1-LacI, these cells form colonies on glucose, but less than 1% form colonies on galactose because they cannot tolerate the loss of chromosome III (). With Ask1 bound to the chromosome, 78 ± 2% of the cells form colonies on galactose plates. This experiment shows that the synthetic kinetochore can segregate a chromosome, although less well than a natural kinetochore, since the colonies are smaller than those of wildtype cells.
To ask how many copies of Ask1 allow chromosome segregation, we repeated this assay with a LacO array containing only 8 repeats. We found that 8 and 256 repeats gave similar results (). Since LacO binds a dimer of LacI, no more than 16 copies of Ask1 can be recruited to the smaller array, similar to the number of Ask1 molecules in the natural kinetochore12
We followed chromosome segregation by expressing a green fluorescent protein-LacI fusion (GFP-LacI) that binds the LacO array13
. This fusion reveals the tagged chromosomes as GFP dots but has no effect on the ability of Ask1-LacI to direct their segregation (data not shown). We monitored chromosome segregation within a single cell cycle by releasing cells from G1, with or without Ask1-LacI, and allowing them to arrest in anaphase (due to the cdc15-2
mutation). In glucose-grown cells, the natural centromere segregated the chromosomes to opposite ends of the spindle in 99±1% of the cells (). With the natural centromere turned off, and without Ask1-LacI, the chromosome tends to stay in the mother cell; only 27±9% segregated their sister chromatids into mother and bud, but 71±9% of cells contained two GFP dots in the mother. With the synthetic kinetochore on, and the natural centromere off, 74±8% of cells segregated sister chromatids properly between mother and bud. This experiment demonstrates that the synthetic kinetochore can direct ordered chromosome segregation, although this segregation is not as faithful as a natural kinetochore.
The synthetic kinetochore can align and segregate chromosomes and correct attachment errors
Natural kinetochores biorient at metaphase; the sister kinetochores attach to opposite spindle poles and are pulled about 0.5μm apart from each other1
. This separation can be seen by placing a GFP tag 1.8kb from the centromere and arresting the cells in metaphase (by depleting Cdc20, the activator of the anaphase promoting complex (APC))14–16
; with the natural centromere active, 74±2% of metaphase-arrested cells contained two separate GFP dots (), whereas the synthetic kinetochore was separated in 55±2% of cells when the natural centromere was off. Without a synthetic kinetochore and with the natural centromere off, only 5±2% of cells contained two GFP dots. We conclude that the synthetic kinetochore can biorient at metaphase.
In some cells, both sister kinetochores initially attach to the same pole of the spindle. Left uncorrected, this error leads to one daughter with two copies of the chromosome and one with none. The kinetochore detects and corrects this error: the protein kinase Ipl1 (the yeast Aurora B homolog) induces kinetochores that are not under tension to release their microtubules17–20
We asked if the synthetic kinetochore could detect and correct orientation errors. Cells carrying the ipl1-321
mutation detect and correct errors at 23 °C but not 37 °C17
. When the mutant went from G1 to metaphase at 23°C, a natural kinetochore was visibly bioriented in 80±2% of the cells (). But if the cells went from G1 to metaphase at 37°C only 23±5% of the cells contained two GFP dots. These errors could be corrected; shifting the metaphase-arrested cells from 37 °C to 23 °C, raised the fraction of cells containing two GFP dots to 76±3%.
We showed that the synthetic kinetochore can also correct errors in attachment (). Cells carrying ipl1-321 and a chromosome with both the galactose-repressible (PGAL1-CEN3) and the synthetic kinetochore were arrested in G1, exposed to galactose to disrupt the formation of the natural kinetochore and allowed to progress from G1 to a metaphase arrest at different temperatures. When Ipl1 was active (23 °C), 51±4% of the synthetic kinetochores were visibly bi-oriented. In contrast, only 21±3% of cells that had reached metaphase at 37 °C contained two GFP dots. When these arrested cells were transferred to 23 °C this fraction rose to 50±2%, showing that the synthetic kinetochore can both sense and correct orientation errors.
Finally, we determined which other kinetochore proteins were required to produce a synthetic kinetochore. We obtained cells with temperature-sensitive mutations in a variety of kinetochore proteins3
and verified that they formed spindles (demonstrating that they had microtubules) and prevented natural kinetochores from biorienting at 37 °C. We asked whether the corresponding proteins were needed to align the synthetic kinetochores on the spindle: if the protein plays no role, the kinetochores will biorient and resolve into two GFP dots at 37 °C, but mutations that affect the synthetic kinetochore will prevent biorientation.
The synthetic kinetochore requires proteins in several different kinetochore complexes. The Ndc80 complex may have the kinetochore’s principal microtubule binding activity1, 21
and the Mtw1 complex appears to regulate this connection14
. We tested mutations in three components of the Ndc80 complex (Ndc80, Nuf2, Spc24) and three of the Mtw1 complex (Nsl1, Dsn1 and Mtw1) and found that all six proteins were needed for biorientation ().
The synthetic kinetochore requires many components of natural kinetochores
We suspected that other members of the Dam1-DASH complex were needed at the synthetic kinetochore. Tests on Dam1 supported this idea; when Dam1 was inactivated, the synthetic kinetochore could no longer biorient. We tested one of the two essential members of the Ctf19 complex3
(Okp1) and found that it is also involved in biorientation of the synthetic kinetochore.
The proteins that bind to centromeric DNA are poorly conserved during evolution. We tested two of four components of CBF3 (Ndc10 and Ctf13), the essential DNA-binding complex of the yeast kinetochore; neither was needed to biorient the synthetic kinetochore () but both mutations completely disrupt biorientation of natural kinetochores (). Our functional analysis suggests that the synthetic kinetochore recruits four complexes, including two that bind microtubules, but does not need the primary sequence-specific DNA binding activity of the natural kinetochore ().