Kinetic framework of spindle assembly checkpoint signalling

Abstract

The mitotic spindle assembly checkpoint (SAC) delays anaphase onset until all chromosomes have attached to both spindle poles 1,2 . Here, we investigated SAC signaling kinetics in response to acute detachment of individual chromosomes using laser microsurgery. Most detached chromosomes delayed anaphase until they had realigned to the metaphase plate. A substantial fraction of cells, however, entered anaphase in the presence of unaligned chromosomes. We identify two mechanisms by which cells can bypass the SAC: First, single unattached chromosomes inhibit the anaphase promoting complex/cyclosome (APC/C) less efficiently than a full complement of unattached chromosomes. Second, because of the relatively slow kinetics of reimposing APC/C inhibition during metaphase, cells were unresponsive to chromosome detachment up to several minutes before anaphase onset. Our study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes. Detailed knowledge about SAC signaling kinetics is important for understanding the emergence of aneuploidy and the response of cancer cells to chemotherapeutics targeting the mitotic spindle.Spindle assembly is an error prone process, which can involve multiple rounds of microtubule attachment and detachment on individual kinetochores 3,4 . Incorrectly attached chromosomes, for example when both sister kinetochores bind to the same spindle pole, are detected by an error correction machinery, which depolymerizes microtubules on kinetochores if they are under low mechanical tension (reviewed in 5 ). This enables chromosomes to eventually attach their sister kinetochores to microtubules originating from opposing spindle poles.Due to the stochastic nature of spindle assembly, anaphase must be robustly delayed when individual chromosomes fail to attach properly. Indeed, a single unattached kinetochore can delay anaphase onset for up to several hours in PtK 1 cells 6 . Yet, even when all kinetochores are unattached by drug-induced depolymerization of microtubules, cyclin B continues to be degraded by low residual APC/C activity, which leads to mitotic exit in presence of an * Address correspondence to DWG. daniel.gerlich@imba.oeaw.ac.at. Author contributions: A.E.D. designed and conducted experiments and analyzed data. D.W.G. conceived the project, analyzed data, and wrote the manuscript with help by A.E.D. Competing financial interests:The authors declare no competing financial interests. [7][8][9] . Mitotic slippage occurs at different rates in a variety of cancer and non-cancer cells and it has been proposed as a mechanism by which cancer cells may develop resistance against therapeutics that target the mitotic spindle [8][9][10][11][12] . Hence, it is important to better understand the signaling kinetics underlying SAC-mediated APC/C inhibition. Europe PMC Funders GroupUnattached kinetochores recruit the SAC protein Mad2 to promote assembly of cytoplasmic mitotic checkpoint complex, composed of Ma...

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