Loss of Function of the Cik1/Kar3 Motor Complex Results in Chromosomes with Syntelic Attachment That Are Sensed by the Tension Checkpoint

Abstract

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.

Premature Silencing of the Spindle Assembly Checkpoint Is Prevented by the Bub1-H2A-Sgo1-PP2A Axis in Saccharomyces cerevisiae

“…The cosegregation of CEN4-GFP in bub1-DK cells indicates checkpoint arrest failure. Our previous study showed that overexpression of CIK1-CC in sgo1D cells caused .30% CEN4-GFP missegregation using the same analysis (Jin et al 2012), which is higher than that of bub1-DK cells. One explanation is that Sgo1 is still functional enough in bub1-DK cells to prevent SAC silencing, but less efficient due to the failure of centromere localization.…”

“…We showed that Ipl1 kinase and Sgo1 are required to prevent SAC silencing in response to syntelic attachment induced by CIK1-CC overexpression (Jin et al 2012;Jin and Wang 2013). Because the centromere localization of Sgo1 depends on H2A phosphorylation by Bub1 kinase (Fernius and Hardwick 2007;Kawashima et al 2010;Nerusheva et al 2014), Bub1 kinase activity might be required to prevent SAC silencing in response to syntelic attachment, although this kinase activity is dispensable for SAC activation (Fernius and Hardwick 2007).…”

“…Previous studies indicate that Sgo1 recruits Ipl1/Aurora B kinase complex to centromeres Peplowska et al 2014). Because Ipl1 kinase prevents SAC silencing in response to tension defects (Biggins and Murray 2001;Jin et al 2012;Jin and Wang 2013), one speculation is that Sgo1 prevents SAC silencing through Ipl1 kinase. However, the observation that the tension sensing by Ipl1 is independent of the centromere localization of Ipl1 complex argues against this possibility (Campbell and Desai 2013).…”

“…The yeast cell growth, synchronization, and CIK1-CC overexpression were performed as described previously (Jin et al 2012). Briefly, yeast cells with a control vector or P GAL CIK1-CC plasmid were grown in synthetic medium containing raffinose to midlog phase and then arrested in G 1 phase with a factor.…”

“…Therefore, this mechanism links bipolar attachment and SAC silencing (Wang et al 2014). SGO1 encodes a centromere-binding protein (Indjeian et al 2005), and SGO1 deletion also leads to premature SAC silencing (Jin et al 2012;Jin and Wang 2013). However, it is largely unknown how Sgo1 regulates SAC silencing at the molecular level.…”