Sli15(INCENP) dephosphorylation prevents mitotic checkpoint reengagement due to loss of tension at anaphase onset

Abstract

The mitotic checkpoint, also known as the spindle assembly checkpoint, delays anaphase onset until all chromosomes have reached bipolar tension on the mitotic spindle [1-3]. Once this is achieved, the protease separase is activated to cleave the chromosomal cohesin complex, thereby triggering anaphase. Cohesin cleavage releases tension between sister chromatids, but why the mitotic checkpoint now remains silent is poorly understood. Here, using budding yeast as a model, we show that loss of sister chromatid cohesion at anaphase onset would engage the mitotic checkpoint if this was not prevented by concomitant separase-dependent activation of the Cdc14 phosphatase. Cdc14, in turn, inactivates the mitotic checkpoint by dephosphorylating Sli15(INCENP), a subunit of the conserved Aurora B kinase complex that forms part of the proposed chromosomal tension sensor. Dephosphorylation-dependent relocation of Sli15(INCENP) from centromeres to the central spindle during anaphase is seen in organisms from yeast to human [4-8]. Our results suggest that Sli15(INCENP) dephosphorylation is part of an evolutionarily conserved mechanism that prevents the mitotic checkpoint from reengaging when tension between sister chromatids is lost at anaphase onset.

Figure 1

Cdc14 Prevents Mitotic Checkpoint Engagement Due to Loss of Sister Chromatid Cohesion at Anaphase Onset (A) Cells were arrested in metaphase by Cdc20 depletion, and expression of separase, TEV protease, or TEV protease together with Cdc14 was induced. Activation of the mitotic checkpoint was monitored by the phosphorylation-induced electrophoretic mobility shift of Mad1, fused to a HA-epitope tag to facilitate western detection. The same cells treated with the spindle poison nocodazole (5 μg/ml; noc), but uninduced, served as a positive control for mitotic checkpoint activation. (B) As in (A), but checkpoint activation was visualized by the appearance of Bub1-GFP nuclear foci. Images are of cells 45 min after induction; scale bar represents 5 μm. Anaphase spindles of 4 μm or longer were scored as elongated. See also Figure S1.

Figure 2

Persistent Mitotic Checkpoint Signaling in cdc14-1 Mutant Anaphase Cells (A) Cells of the indicated genotypes were released from α-factor block in G1 into synchronous cell cycle progression at nonpermissive temperature (37°C) for the cdc14-1 and cdc15-2 alleles. Cell cycle progression was monitored by fluorescence-activated cell sorting (FACS) analysis of DNA content. (B) Spindles of 4 μm or longer were scored as elongated. (C) The Mad1 phosphorylation status in cells from the above experiment was analyzed by western blotting. (D) Levels of securin (Pds1), fused to a myc epitope tag to facilitate detection, were analyzed by western blotting. Tubulin served as a loading control. See also Figure S2.

Figure 3

Cdc14 Relieves the Mitotic Checkpoint Delay Due to Absence of Tension (A and B) Wild-type (A) and scc1-73 (B) cells were grown in YP medium containing raffinose as carbon source, arrested in G1 using α-factor, and released into synchronous cell cycle progression at restrictive temperature (35°C). α-factor was added back at 75 min for rearrest in the following G1. Cell cycle progression was monitored by FACS analysis of DNA content and western blotting against securin (Pds1) fused to a HA-epitope tag. Cdc28 served as a loading control. (C) In a second scc1-73 culture, Cdc14 expression from the GAL1 promoter was induced by galactose addition at 60 min. (D) A third scc1-73 culture carried the sli15-6A allele. See also Figure S3.

Figure 4

Nonphosphorylatable Sli15-6A Prevents Mitotic Checkpoint Reengagement in Anaphase (A) Budding yeast cells harboring wild-type SLI15 or the sli15-6A allele were arrested in metaphase by Cdc20 depletion. Loss of sister chromatid cohesion was triggered by TEV protease expression. Mad1 phosphorylation and anaphase spindle elongation were monitored as in Figure 1. (B) Model for mitotic checkpoint inactivation in anaphase. During chromosome alignment on the mitotic spindle in prometaphase, a mitotic checkpoint signal, including the Mad1 and Bub1 proteins, emanates from kinetochores that have not yet come under tension. This prevents APC activation by Cdc20. Generation of the checkpoint signal depends on the physical proximity between the Aurora B kinase complex and its targets on tensionless kinetochores. Once bipolar tension is established in metaphase, the checkpoint is silenced and the APC degrades securin to activate separase. Cohesin cleavage now triggers anaphase, and tension is lost again from kinetochores. This would reactivate the checkpoint, but this is prevented by Sli15^INCENP dephosphorylation and consequent relocation of the Aurora B kinase complex to the spindle midzone. Dephosphorylation of additional Cdk targets might contribute to maintain an inactive checkpoint. See also Figure S4.