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Abstract

The spindle assembly checkpoint (SAC) is a surveillance mechanism, which ensures that cells only enter anaphase once all chromosomes have become properly attached to the mitotic spindle. The proteins constituting the SAC network are known and are conserved in eukaryotes. SAC proteins enrich at unattached kinetochores and ultimately lead to the inhibition of the anaphase-promoting complex. However, the complex in vivo signaling pathway is still only fragmentarily understood. Mathematical modeling can be a valuable means to explore possibilities for the signaling mechanism, but requires accurate quantitative data. We have determined the relative and absolute abundance of GFP-labeled SAC proteins in vivo in fission yeast by quantitative fluorescence microscopy and fluorescence correlation spectroscopy. The SAC proteins differ in their abundance, but all are present in the fission yeast cell at low nanomolar concentration. To further explore the robustness of the signaling mechanism and our mathematical representation of it, we modified SAC protein abundances and compared the outcome on SAC activity. We found strong differences in the ability of the SAC to tolerate changes in the abundance of single SAC proteins, and these results shed light on the signaling mechanism. To further unravel the organization of the SAC network, we determined the hierarchy of SAC protein localization to kinetochores upon mitotic entry and SAC activation. When comparing our results to data obtained in other eukaryotes, evolutionary differences become apparent. Exploring how the conserved SAC signaling mechanism has been shaped in different organisms in adaptation to their specific needs will be an interesting area for future investigation.