Ska3 Is Required for Spindle Checkpoint Silencing and the Maintenance of Chromosome Cohesion in Mitosis

Abstract

SUMMARY Balanced chromosome segregation in mitosis requires synchronous chromatid separation at anaphase and the precise coordination of anaphase with cytokinesis and mitotic exit. The mitotic spindle checkpoint monitors proper attachment and/or tension induced by microtubule binding to sister kinetochores. Within each cell, once all chromosomes achieve bipolar attachment to the spindle poles and align at the metaphase plate, the spindle checkpoint is silenced triggering anaphase onset, cytokinesis, and mitotic exit. We used a bioinformatics approach to identify a candidate protein, C13orf3/Ska3, predicted to function in mitosis. Cells in which Ska3 expression was reduced by RNAi achieved metaphase alignment but were unable to silence the spindle checkpoint and enter normal anaphase. After hours of metaphase arrest, chromatids separated but retained robust attachment to spindle microtubules. These cells remained checkpoint arrested with strong accumulation of the checkpoint protein Bub1 at kinetochores. During normal mitosis Ska3 protein accumulated on kinetochores in prometaphase after nuclear envelope breakdown. This kinetochore localization of Ska3 was dependent on Shugoshin (Sgo1), the “guardian spirit” of chromatid cohesion. In contrast, Sgo1, which accumulates at the centromeres in early prophase, was not dependent on Ska3. Although Ska3 is required for maintenance of sister chromatid cohesion and is dependent upon Sgo1, cells with reduced Sgo1 show a stronger premature chromatid separation phenotype than those with reduced Ska3. We hypothesize that Ska3 functions as a component of a network that coordinates checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the spindle checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.

The KMN protein network – chief conductors of the kinetochore orchestra

“…SKA1 and SKA2 were found to localize to spindle MTs and to concentrate at kinetochores in a manner that is dependent on kinetochorebound NDC80 (Hanisch et al, 2006). Subsequently, a third subunit of the complex, SKA3, also called RAMA1, was discovered that localizes to kinetochores and spindle MTs, and knockdown of this protein results in identical phenotypes to those upon knockdowns of SKA1 and SKA2 (Daum et al, 2009;Gaitanos et al, 2009;Raaijmakers et al, 2009;Theis et al, 2009;Welburn et al, 2009). SKA3 is required for the proper stability and functioning of the Ska complex as a whole (Gaitanos et al, 2009).…”

The dynamic protein Knl1 – a kinetochore rendezvous

“…KMN network proteins (although not Knl1 itself) have also been shown to directly interact with the spindle and kinetochoreassociated (SKA) protein complex formed by SKA1, SKA2 and SKA3, which has been strongly implicated in maintaining stable kinetochore-MT interactions (Hanisch et al, 2006;Gaitanos et al, 2009;Theis et al, 2009;Daum et al, 2009;Raaijmakers et al, 2009). The association between the KMN and SKA networks is negatively regulated by Aurora B activity (Chan et al, 2012).…”

The Ndc80 internal loop is required for recruitment of the Ska complex to establish end-on microtubule attachment to kinetochores.

“…This includes the spindle and kinetochoreassociated (Ska) complex (Daum et al, 2009;Gaitanos et al, 2009;Hanisch et al, 2006;Theis et al, 2009;Welburn et al, 2009), the checkpoint proteins Bub1 (Meraldi and Sorger, 2005) and BubR1 (Lampson and Kapoor, 2005), Cenp-E (Yen et al, Fig. 1.…”