Bub1 availability limits the Spindle Assembly Checkpoint signaling strength of human kinetochores

Abstract

During mitosis, unattached kinetochores in a dividing cell signal to the Spindle Assembly Checkpoint to delay anaphase onset and prevent chromosome missegregation 1-4. The signaling activity of these kinetochores and the likelihood of chromosome missegregation both depend on the amount of SAC signaling proteins that each kinetochore recruits 5-8. Therefore, factors that control SAC protein recruitment to signaling kinetochores must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins emerging from the concerted action of many kinases and phosphatases is a major determinant of SAC protein recruitment to signaling kinetochores 9. Whether the abundance of SAC proteins also influences their recruitment and signaling activity at human kinetochores has not been studied 8, 10. Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits kinetochore recruitment of Bub1 and BubR1 and reduces the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher protein recruitment and SAC activity producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins. The inverse correlation between the number of signaling kinetochores in the cell and the signaling activity of individual kinetochores may allow the dividing cell to prevent the large number of signaling kinetochores in prophase from generating an unnecessarily large signal, while enabling the last unaligned kinetochore to signal at the maximum possible strength.