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Abstract

The transition from metaphase to anaphase is one of the crucial steps in the cell cycle, at which the events of sister chromatid separation and mitotic exit need to be precisely coordinated. Central to the transition is the anaphase promoting complex/cyclosome (APC/C), which targets cyclin B and securin for proteasomal destruction. Cyclin B degradation inactivates the cyclin- dependent kinase CDK1 and hence promotes mitotic exit. Securin degradation releases its binding partner separase from the inhibitory association. Subsequently, free separase proteolytically cleaves a cohesin subunit and thereby promotes the loss of chromosomal cohesion and the irreversible separation of sister chromatids. Surprisingly, sister chromatid separation is highly synchronous and about 20 times faster than the underlying securin degradation. We want to understand how slow securin degradation translates into abrupt sister chromatid separation and how this contributes to the robust coordination of the events during mitotic exit. To this end, we employ a combination of live cell imaging, quantitative proteomics and computational modeling. We have established two imaging assays with which we follow securin degradation and sister chromatid separation at high temporal resolution in Schizosacharomyces pombe. Consistent with findings in other organisms, we show that in S. pombe securin degradation is about a magnitude slower than sister chromatid separation. Furthermore, sister chromatid separation occurs invariantly when 25 % of securin have been degraded. In order to approach the underlying mechanism of this ‘switch’-like behavior, we systematically changed abundances or activities of key proteins at the transition and observed the system’s response to the alterations. First results suggest that APC/C but not CDK1 activity influences the threshold level at which sister chromatid separation takes place as well as the ‘sharpness’ of sister chromatid separation. We have subsequently tested several models of the transition, e.g. inhibitor- sequestration or positive feedback models, in silico for their ability to reconcile with our experimental data. Currently, we are in the process of testing experimentally the predictive power of the different models by further system changes.