Elucidating the role of Bub1 in human checkpoint signalling

The spindle assembly checkpoint (SAC) establishes a delay before anaphase, until all chromosomes are equally and stably attached to spindle microtubules via kinetochores. This acts as a surveillance mechanism to prevent chromosome segregation defects and safeguard genomic stability. The exact mechanisms regulating checkpoint activation and mitotic progression remain unclear. Previous work has shown that SAC activation requires Mps1-dependant phosphorylation of KNL1 MELT motifs. This allows recruitment of Bub1-Bub3, and subsequent localisation of BubR1-Bub3 and Mad1/Mad2 complexes onto Bub1. Localisation of Mad1/Mad2 to the kinetochore allows production of the mitotic checkpoint complex (MCC), which inhibits the APC/C to prevent initiation of anaphase. This was thought to represent a single, linear pathway for SAC activation. However, recent work in human cells has provided evidence that the Rod-Zwilch-ZW10 (RZZ) complex can offer a second receptor for Mad1/Mad2 complexes, thus allowing two separable pathways for SAC activation and maintenance in human cells. We have tested this dual-pathway model by knocking out BUB1 in human hTERT-RPE1 cells using CRISPR-Cas9 technology. These Bub11-23 cells are able to generate a functional checkpoint in the presence of unattached kinetochores, however, Bub11-23 cells were later found to express residual amounts of Bub1 protein, referred to as ‘zombie’ Bub1. We have therefore repeated all key experiments using Bub11-23 cells in the presence of Bub1 siRNA to deplete ‘zombie’ Bub1, and again shown that these cells are able to generate a functional checkpoint in the presence of unattached kinetochores, although maintenance is perturbed. Furthermore, we have found that Bub1 plays an important role in the regulation of unperturbed mitosis which is at least partially mediated through an uncharacterised premitotic function. Together, these data provide further evidence for a Bub1-independent mechanism of checkpoint activation at unattached kinetochores, and uncover a novel role for Bub1 during interphase.