Tension sensing by Aurora B kinase is independent of survivin-based centromere localization

Abstract

Accurate segregation of the replicated genome requires chromosome biorientation on the spindle. Biorientation is ensured by Aurora B kinase (Ipl1), a member of the four-subunit chromosomal passenger complex (CPC). Localization of the CPC to the inner centromere is central to the current model for how tension ensures chromosome biorientation: kinetochore-spindle attachments that are not under tension remain close to the inner centromere and are destabilized by Aurora B phosphorylation, whereas kinetochores under tension are pulled away from the influence of Aurora B, stabilizing their microtubule attachments. Here we show that an engineered truncation of the Sli15 (known as INCENP in humans) subunit of budding yeast CPC that eliminates association with the inner centromere nevertheless supports proper chromosome segregation during both mitosis and meiosis. Truncated Sli15 suppresses the deletion phenotypes of the inner-centromere-targeting proteins survivin (Bir1), borealin (Nbl1), Bub1 and Sgo1 (ref. 6). Unlike wild-type Sli15, truncated Sli15 localizes to pre-anaphase spindle microtubules. Premature targeting of full-length Sli15 to microtubules by preventing Cdk1 (also known as Cdc28) phosphorylation also suppresses the inviability of Bir1 deletion. These results suggest that activation of Aurora B kinase by clustering either on chromatin or on microtubules is sufficient for chromosome biorientation.

Figure 1. Deletion of the Sli15 N-terminus prevents association with Bir1 but does not affect cell viability or growth

(a) Schematic of the CPC in budding yeast. Sli15=INCENP; Bir1=Survivin; Nbl1=Borealin/Dasra and Ipl1=Aurora B kinase. (b) Phenotype of bir1Δ. Tetrad dissections from a bir1Δ heterozygote—the 4 spores from individual tetrads are arrayed in columns. Rare survivors (referred to as bir1Δ*) are observed after extended growth (right panel). (c) Phenotype of Sli15 truncations. Tetrad dissections from a sli15ΔNT heterozygote are shown on the right. (d) Co-immunoprecipitation analysis of full length (FL) Sli15 or Sli15ΔNT and Bir1. 9Myc and 6HA tags were inserted at endogenous loci to generate functional C-terminally tagged proteins. Bir1 coimmunoprecipitates with FL Sli15 but not with Sli15ΔNT. (e) Localization of Sli15-Venus and Sli15ΔN-Venus to the anaphase spindle. Scale bar is 5 μm. (f) Localization of CPC components during anaphase in cells with either wildtype Sli15 or Sli15ΔNT. Scale bar is 5 μm.

Figure 2. sli15ΔNT suppresses lethality of bir1Δ and nbl1Δ, and exhibits normal fidelity mitotic and meiotic chromosome segregation

(a) Tetrad dissection showing viability of sli15ΔNT;bir1Δ double mutant spores. (b) 10-fold serial dilutions of cells of indicated genotypes. bir1Δ* and nbl1Δ* represent rare survivors recovered as shown in Fig. 1B. (c) Immunoblot of extracts prepared from strains expressing either wild type Sli15 or Sli15ΔNT and blotted using an antibody raised against the C-terminus of Sli15. The asterisk (*) indicates a non-specific band recognized by the primary antibody that serves as a loading control. (d) Analysis of segregation fidelity of GFP-tagged chromosome IV in cells of the indicated genotypes. The average of 3 to 5 experiments is shown; error bars represent standard error. (e) Minichromosome loss assay. The percentage and standard error of colonies that were either red or sectored is shown. (f) Viability following transient nocodazole treatment of cells of the indicated genotypes. The average of 2 to 4 experiments is shown; error bars represent standard error. (g) Serial dilutions of cells with the indicated genotypes spotted on plates with different concentrations of the microtubule-depolymerizing drug benomyl. (h) Lack of synthetic lethality/sickness following checkpoint inhibition in sli15ΔNT cells. Plates were incubated at 37°C. sli15-3 is a temperature-sensitive mutant that compromises Ipl1 activation. (i) Meiotic segregation following sporulation of diploid cells. The presence or absence of Chromosome I was scored for each individual spore in a tetrad. Scale bar is 5 μm.

Figure 3. sli15ΔNT suppresses mutants in the Bir1-dependent CPC targeting pathway but is synthetically lethal with genes implicated in centromere cohesion

(a) Schematic of the CPC targeting mechanism involving Bub1 kinase and Sgo1. (b) Suppression of sgo1Δ and bub1Δ growth phenotypes by sli15ΔNT. Compromised growth of sgo1Δ on benomyl is also suppressed by sli15ΔNT. (c) Viability following transient nocodazole treatment of cells of the indicated genotypes. The average of 2 to 4 experiments is shown; error bars represent standard error. WT and sli15ΔNT measurements are the same as in Fig. 2f. (d) Tetrad analysis showing no synthetic defect for cells that are triple mutants for sli15ΔNT alk1Δ and alk1Δ. (e) Tetrad analysis showing synthetic lethality of sli15ΔNT and mcm21Δ. Similar results were observed for ctf19Δ (Fig. S2b). (f) Summary of genetic interactions exhibited by sli15ΔNT. A positive genetic interaction (green) indicates suppression; negative genetic interaction (red) indicates synthetic lethality/sickness and neutral interaction (black) indicates lack of a synthetic phenotype.

Figure 4. Localization of Sli15ΔNT and relationship between Sli15 microtubule localization and suppression of bir1Δ

(a) Images of cells expressing Nuf2-mCherry and either Sli15-Venus or Sli15ΔNT-Venus after brief microtubule depolymerization. A cell with ~1 μm separation of Nuf2-mCherry clusters is shown for each. Boxes are 2.1 μm square. 4 additional examples are shown on the right. See Fig. S3A for similar analysis of Ipl1 localization. (b) Images of cells arrested in metaphase by Cdc20 depletion expressing Nuf2-mCherry and either Sli15-Venus or Sli15ΔNT-Venus. Scale bar is 5 μm; merged insets are magnified 2.5-fold. See Fig. S3B for similar analysis of of Ipl1 localization. (c) Schematic summarizing prior work on Cdk1 regulation of Sli15 spindle localization and tetrad analysis showing growth of sli15-6A;bir1Δ double mutant cells. (d) Model for mechanism of chromosome biorientation. Bir1-dependent chromatin clustering of the CPC or Bir1-independent clustering on microtubules of Sli15ΔNT-Ipl1 generates active Ipl1 kinase, which is capable of discriminating between correct and incorrect attachments.