The kinetochore clustering in budding yeast has been documented for many years, but the biological significance of this clustering has remained a mystery. Moreover, it is unclear whether this clustering is totally dependent upon the KT-MT interaction. Here we report that the kinetochore protein Slk19 clusters kinetochores, which is likely independent of KT-MT interaction. We further show that, in the absence of Slk19, kinetochore capture and the establishment of chromosome bipolar attachment are delayed after the disruption of the KT-MT interaction by nocodazole. We also show in vivo and in vitro evidence indicating that Slk19 protein interacts with itself. Therefore we propose a model in which Slk19–Slk19 interaction leads to kinetochore clustering (), a mechanism that becomes critical for chromosome capture and bipolar attachment after the disruption of KT-MT interaction.
Our data indicate that the Slk19-mediated kinetochore clustering is likely independent of the microtubules that connect kinetochores to the spindle poles. First, the scattered kinetochores in nocodazole-treated slk19
Δ mutant cells are away from the spindle pole, suggesting the loss of connection with the spindle poles. Moreover, the slk19
Δ mutant cells show similar microtubule depolymerizing kinetics as WT cells in response to nocodazole treatment (). In addition to spindle poles, recent data indicate that kinetochores also generate microtubules (Ortiz et al., 2009
; Kitamura et al., 2010
). Thus it is possible that Slk19 promotes kinetochore clustering by associating and/or stabilizing these microtubules. Another possibility is that Slk19 binds to other kinetochore proteins directly, and the Slk19–Slk19 interaction leads to kinetochore clustering.
Previous work showed abnormal nuclear morphology in slk19
Δ mutant cells (Zhang et al., 2006
). One interesting question is whether the kinetochore clustering defect is secondary to the abnormal chromatin structure. Because nocodazole treatment abolishes the abnormal nuclear morphology seen in slk19
Δ cells (Zhang et al., 2006
), the kinetochore declustering in nocodazole-treated slk19
Δ cells is unlikely a consequence of the abnormal nuclear morphology. Instead, the enriched localization of Slk19 at kinetochores suggests that the kinetochore function of Slk19 likely contributes to kinetochore clustering. In addition, the data from the Dawson lab show the colocalization of Slk19 with another kinetochore protein, Mtw1 (Havens et al., 2010
). We also performed live-cell imaging in cells with SLK19-GFP NUF2-mCherry
and found the colocalized Slk19 and kinetochore protein Nuf2 until anaphase, when the spindle midzone localization of Slk19 was noticed (unpublished data). Because we showed direct Slk19–Slk19 interaction, one possibility is that the interaction between Slk19 proteins from different kinetochores mediates kinetochore clustering (), but more experiments are needed to confirm this model.
In nocodazole-treated slk19Δ mutant cells, in addition to a major kinetochore cluster that associated with the spindle poles, more small Mtw1-GFP foci were detected. One interesting question is whether the smaller Mtw1-GFP dot represents a single pair of sister kinetochores. In some slk19Δ cells, we observed as many as nine Mtw1-GFP foci. Given that yeast cells have only 16 pairs of sister chromatids in G2/M phase, some of the smaller foci have to be a single pair of sister kinetochores. In nocodazole-treated slk19Δ cells, some kinetochores may stay together even when they are disconnected from the spindle pole. One possibility is that these kinetochores just colocalize by chance but without any linkage. Alternatively, an unknown mechanism clusters these kinetochores in the absence of Slk19 and KT-MT interaction. Therefore further analysis is necessary to determine whether kinetochore clustering is abolished completely in slk19Δ mutant cells once they become disconnected from the spindle pole.
Kinetochore declustering was also observed in other kinetochore mutants, such as nuf2-60
, even without treatment with nocodazole (Janke et al., 2001
; Anderson et al., 2009
). Nuf2 and Ndc80 are components of the Ndc80 kinetochore complex, which is required for the KT-MT interaction. Recent data show that the association of Slk19 with kinetochore is abolished in some temperature-sensitive kinetochore mutant strains when incubated at 37°C, including spc105ts
, and mtw1-1
(Pagliuca et al., 2009
). Therefore it is possible that the abolished KT-MT interaction alone causes kinetochore clustering. Alternatively, both the loss of the KT-MT interaction and the absence of Slk19 at kinetochores are necessary for kinetochore declustering.
What is the biological significance of this Slk19-dependent kinetochore clustering? For the kinetochores that lose their connection to the spindle pole, the highly dynamic spindle microtubules are responsible for their capture. Then the minus end–directed motor complex Cik1/Kar3 transports the captured kinetochores toward the vicinity of the spindle pole (Tanaka et al., 2005
). The increased distance between a kinetochore and the spindle pole will significantly reduce the chance of kinetochore capture. A whole kinetochore cluster will be moved to the spindle pole once only one kinetochore in this cluster is captured. This mechanism is expected to increase the efficiency for the transport of detached kinetochores toward the vicinity of a spindle pole, where they have a much higher chance to be captured by spindle microtubules. In support of this speculation, we observed a dramatic kinetochore capturing delay in slk19
Δ mutant cells after the disruption of KT-MT interaction. Therefore kinetochore clustering could be an unidentified mechanism that facilitates kinetochore capture.
In budding yeast, an interesting observation is that sister kinetochores separate before anaphase entry (Goshima and Yanagida, 2000
; He et al., 2000
). Because separase is inactive before anaphase onset, cohesin cleavage is unlikely the cause for this sister centromere separation. Work from the Bloom lab showed a cruciform structure of the pericentric chromatin (Yeh et al., 2008
). This observation indicates that the enriched cohesin at the pericentric region unlikely links sister centromeres, and instead it might stabilize the cruciform structure by introducing cohesion within a single chromatin at pericentric regions. Thus a unique mechanism may contribute to sister chromatid cohesion at centromeric regions. Our evidence suggests that Slk19 mediates interaction between kinetochores from different chromosomes. It is possible that Slk19 also mediates interaction between sister kinetochores, which contributes to sister-chromatid cohesion at centromeric regions. To test this possibility, we looped out a pair of GFP-marked centromeres of chromosome IV in nocodazole-treated cells and found that slk19
Δ cells showed higher frequency of separated sister centromeres (Supplemental Figure S8). However, the result could not support a solid conclusion because of the lower frequency of the excision of the centromere from chromosome IV, as indicated by only 40% viability loss after the induction of recombination. If the Slk19–Slk19 interaction contributes to sister kinetochore cohesion, the tension resulting from chromosome bipolar attachment can separate sister kinetochores and centromeres by breaking up the Slk19-Slk19 interaction in the absence of separase-dependent cohesin cleavage. Moreover, this Slk19-mediated sister centromere cohesion should be reversible. Indeed, separated sister centromeres in metaphase cells reunite after nocodazole treatment, and this reunion depends on Slk19 (Zhang et al., 2006
). Therefore both kinetochore clustering and sister chromatid cohesion at centromere regions could be attributed to Slk19-mediated interkinetochore interaction ().
Our data also indicate the role of Slk19 in the establishment of chromosome bipolar attachment. The exposure of cdc13-1 slk19Δ cells to nocodazole delayed the chromosome bipolar attachment significantly after nocodazole was washed off, and this delay depends on a functional tension checkpoint, indicating the presence of syntelic attachments. Because we did not observe obvious kinetochore declustering in these cells, it is likely that Slk19 also facilitates chromosome bipolar attachment in a kinetochore-clustering–independent manner. One explanation is that the Slk19–Slk19 interaction between sister kinetochores ensures their opposite orientation, which facilitates the bipolar attachment. In the absence of Slk19, however, the presence of misorientated sister kinetochores increases the frequency of syntelic attachment, especially after the disruption of KT-MT interaction.
Kinetochore clustering is also observed in higher eukaryotes (Solovei et al., 2004
), indicating that this mechanism could be conserved. Although a Slk19 homologue is lacking in higher organisms based upon protein sequence homology, several functional orthologues have been suggested due to its diverse functions (Sato et al., 2003
; Vos et al., 2006
; Ohkuni et al., 2008
). One of the candidates is the mammalian protein CENP-F/mitosin, which localizes to the kinetochore from late G2
to early anaphase but moves to spindle midzone in late anaphase, a pattern similar to that of Slk19 (Rattner et al., 1993
; Liao et al., 1995
; Zhu et al., 1995
; Yang et al., 2003
). Of interest, knockdown of CENP-F leads to weakened centromeric-specific cohesion (Holt et al., 2005
). Moreover, more-scattered chromosome distribution in CENP-F knockdown cells is consistent with the function of CENP-F in kinetochore clustering, although further experiments are needed to confirm this notion (Holt et al., 2005
). Similar to Slk19, CENP-F has also been shown to dimerize (Zhu et al., 1995
). These similarities suggest that CENP-F could be the functional orthologue of yeast Slk19 in mammalian cells.