Chromosome bi-orientation on the mitotic spindle

Abstract

For proper chromosome segregation, sister kinetochores must attach to microtubules extending from opposite spindle poles prior to anaphase onset. This state is called sister kinetochore bi-orientation or chromosome bi-orientation. The mechanism ensuring chromosome bi-orientation lies at the heart of chromosome segregation, but is still poorly understood. Recent evidence suggests that mal-oriented kinetochore-to-pole connections are corrected in a tension-dependent mechanism. The cohesin complex and the Ipl1/Aurora B protein kinase seem to be key regulators for this correction. In this article, I discuss how cells ensure sister kinetochore bi-orientation for all chromosomes, mainly focusing on our recent findings in budding yeast.

Figure 1

Modes of kinetochore–microtubule interactions. Monotelic attachment: one of the sister kinetochores attaches to microtubules, whereas the other does not attach to any microtubules. Syntelic attachment: both sister kinetochores attach to microtubules extending from one spindle pole. Amphitelic attachment: each sister kinetochore attaches to microtubules extending from opposite spindle poles. Merotelic attachment: a single kinetochore simultaneously attaches to microtubules extending from both spindle poles.

Figure 2

The process of generating an unreplicated dicentric circular minichromosome (Dewar et al. 2004) is illustrated. ARS, DNA replication origin; RS, recombination site; P_MET3, MET3 promoter; P_GAL, GAL1-10 promoter; Met, methionine; Gal, galactose; Glc, glucose. The original minichromosome had two centromeres; one (centromere 1) was constitutively active, and the other (centromere 2) was conditionally inactivated owing to its juxtaposition to the GAL1-10 promoter (Hill & Bloom 1987). The chromosome also possessed a single replication origin flanked by recombination sites for the R recombinase (Matsuzaki et al. 1990). The R recombinase was conditionally expressed from the MET3 promoter that is turned on in the absence of methionine. To visualize the behaviour of this minichromosome, we integrated a tandem array of tet operators that bind Tet repressor–GFP fusion proteins (Michaelis et al. 1997). The minichromosome was stably propagated as long as cells were grown without expression of the R recombinase and the centromere 2 was maintained inactive. To obtain an unreplicated dicentric chromosome, we first removed the replication origin on the minichromosome by expressing the R recombinase. Then, we activated the centromere 2 by turning off the adjacent GAL1-10 promoter.

Figure 3

Reorientation of unreplicated centromeres between the new and the old SPBs. The behaviours of unreplicated centromeres (four representative ones are illustrated) in IPL1+ and ipl1 mutant cells (Tanaka et al. 2002) are shown. To inhibit DNA replication (tension is not applied on unreplicated centromeres), we depleted Cdc6 that is required for DNA replication initiation. The old SPB, inherited from the previous cell cycle, remained intact during SPB duplication (Adams & Kilmartin 2000) and always entered the bud during anaphase (Pereira et al. 2001). The new SPB was formed in the vicinity of the old SPB during S phase and remained in the mother cell during anaphase. In yeast, centromeres are tethered to the old SPB by microtubules in G1 phase (Winey & O'Toole 2001; Tanaka et al. 2002). During anaphase of Cdc6-depleted IPL1+ cells, unreplicated centromeres segregated with the old and new SPBs with equal frequency (Piatti et al. 1995; Stern & Murray 2001). This suggests that, after the new SPB had been formed, kinetochores had detached from the old SPB and reoriented between the old and new SPBs. By contrast, in Cdc6-depleted ipl1 mutant cells, unreplicated centromeres predominantly segregated with the old SPB during anaphase (Tanaka et al. 2002). This implies that the frequency of reorientation from the old SPB to the new SPB is considerably reduced when Ipl1 is defective.

Figure 4

A model on how the Ipl1–Sli15 complex facilitates chromosome bi-orientation. The Ipl1–Sli15 kinase complex facilitates bi-orientation by promoting reorientation of kinetochore–spindle pole connections in a tension-dependent manner (Tanaka et al. 2002; Dewar et al. 2004). Because the syntelic attachment does not generate tension on kinetochore-to-pole connections, the Ipl1–Sli15 complex promotes reorientation of these connections (by phosphorylating kinetochore components; see text). When amphitelic attachment is established, tension is applied on kinetochore-to-pole connections and, thereby, the Ipl1–Sli15 complex stops promoting their reorientation, resulting in preferential selection of the amphitelic attachment. Using a chromatin immunoprecipitation assay and fluorescence microscopy, we and others found that Ipl1 localizes at centromeres similarly in the presence and absence of tension exerted on kinetochores during pre-anaphase (K. Tanaka et al. unpublished; Buvelot et al. 2003). It is still unclear how tension leads to cease of the reorientation.

Figure 5

Orientation of the initial kinetochore-to-pole microtubule connections. The ipl1 mutant cells give important clues to early kinetochore–microtubule interactions that have happened before reorientation of kinetochore-to-pole connections is initiated, because the reorientation is blocked in ipl1 mutant cells. The initial microtubule connections of early- and late-replicating centromeres (Tanaka et al. 2002) are illustrated. Thin and thick lines represent microtubules and chromosomes, respectively. The actual experiments were done using circular minichromosomes. Here, to simplify the model, linear chromosomes are shown where replication is initiated in the vicinity of centromeres. When centromeres replicate in early S phase, mono-orientation of sister kinetochores is predominantly found at the old SPB in ipl1 mutant cells. By contrast, when centromeres replicate in late S phase, mono-orientation is found equally at the old and new SPBs. These results address whether centromere DNA replication abolishes pre-existing attachment to microtubules or not (note that centromeres are tethered to the SPB in G1 phase). Formation of mono-orientation to the new SPB suggests that DNA replication indeed transiently disrupts kinetochore–microtubule interactions (see rectangles).