Mechanisms of Spindle Assembly Checkpoint Silencing

Abstract

The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Over the years, through a combination ofcell biology, evolutionary analysis and top-of-the-nudge biochemical techniques, significant progress has been achieved in unravelling and understanding the molecular mechanisms supporting the SAC. Here, we explore some of the open questions regarding the mechanisms of SAC silencing. We focus on the kinetochore and provide novel insights into how microtubule attachments lead to SAC silencing. We investigate what aspect of chromosome-spindle interactions the SAC actually monitors: the kinetochore-microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? We manipulate the microtubule-binding affinity of the kinetochore to distinguish between these aspects and show that kinetochore-microtubule attachments are sufficient to silence the SAC effectively. To explore the mechanisms that drive attachment-induced silencing, we review and discuss the current views and research on the molecular changes in the kinetochore that are evoked by microtubule attachments. Deep understanding of these processes is essential for unravelling the mechanisms of SAC signalling and silencing. In addition, we argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. To investigate our proposed hypothesis, we investigate if SAC silencing requires maximum microtubule occupancy at kinetochores, and how the various molecular events of SAC activation and silencing respond to intermediate occupancy states. To this end, we combine quantitative analysis of levels of SAC proteins on single kinetochores, with manipulations of the microtubule-binding affinity of the kinetochore. We show that SAC proteins are responsive to intermediate microtubule occupancy states and that they separate in two modules of response. Moreover, we find that half-maximal occupancy is sufficient to remove all SAC proteins from kinetochores and silence the SAC. In the final part of this thesis, we introduce a new model system into the field that potentially could help understand diversity in the biology of mitotic processes. We make an effort to understand the diversity of mitotic processes and structures found throughout evolution. We develop basic assays for studying mitosis in U. maydis, identify and visualize homologs of mitotic proteins, and find protein-protein interactions between known mitotic binding partners. In the future, U. maydis can be used alongside other model species to uncover and understand the diversity of SAC signalling.