Abstract 3868: Long-term inhibition of cells at metaphase with intact mitotic spindles results in unscheduled chromatid separation and catastrophic arrest in mitosis

Abstract

Abstract In the presence of unaligned chromosomes or other spindle abnormalities, the mitotic spindle checkpoint blocks both chromatid separation at anaphase and exit from mitosis. The spindle checkpoint is sustained by signaling from kinetochores that are not properly attached to microtubules of the mitotic spindle. Normally, when chromosomes align at the spindle equator at metaphase, microtubule-kinetochore interactions act to extinguish checkpoint signaling. The cessation of the checkpoint signal activates the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. The APC/C ubiquitylates the mitotic effectors, securin and cyclin B, which are then degraded by the proteasome. Securin and cyclin B degradation initiates chromatid separation at anaphase and triggers mitotic exit. We have found that long-term arrest of Hela cells with their chromosomes aligned at the metaphase plate results in chromatid separation without mitotic exit. Recently, we and others have characterized C13orf3/Ska3, a newly discovered component of the Ska (Spindle and kinetochore-associated) protein complex. We found that in mitosis Ska3 protein accumulates at kinetochores reaching maximal levels at metaphase. Upon anaphase onset, kinetochore-associated Ska3 levels decline and are eventually lost at telophase. We demonstrated that cells depleted of Ska3 by RNAi achieve metaphase alignment but fail to silence the spindle checkpoint. During this mitotic arrest with chromosomes aligned at the metaphase plate, kinetochores retain robust microtubule attachments. Kinetochores within these Ska3-depleted cells accumulate abnormally high levels of the mitotic spindle checkpoint protein, Bub1, presumably accounting for the inability to silence the checkpoint. Under extended metaphase arrest, the chromatids of individual chromosomes eventually separate, but cells remain arrested in mitosis. To test if other means of inducing metaphase arrest would also lead to chromatid separation, we treated cells in mitosis with the proteasome inhibitor, MG132. Again, after several hours at metaphase, chromatids separated. We speculate that for cells arrested at metaphase, continual pulling forces of the kinetochores on the mitotic spindle eventually overcomes the cohesin complex that holds chromatids together. This “cohesin slippage” results in abnormal chromatid separation for cells still in mid-mitosis. This response reflects a novel terminal phenotype for cells arrested in mitosis with an intact mitotic spindle. (Supported by the NIGMS and the McCasland Foundation.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3868.