Dissecting the roles of human BUB1 in the spindle assembly checkpoint

Abstract

Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 (forming APC/C CDC20 ). APC/C CDC20 is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/C CDC20 inhibitor that comprises BUBR1 (also known as BUB1B), BUB3, MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these proteins to the kinetochore, as well as SAC activation, rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that kinetochore recruitment of BUBR1 and BUB3 by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 (also known as MAD1L1) and, although required for accelerating the loading of MAD1 onto kinetochores, BUB1 is dispensable for the maintenance of steady-state levels of MAD1 there. Instead, we identify a 50-amino-acid segment that harbors the recently reported ABBA motif close to a KEN box as being crucial for the role of BUB1 in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not of MAD1 to kinetochores.

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

“…Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1,2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4][5][6][7][8][9]. Through gene editing and livecell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons.…”

“…In contrast, studies in mammalian cells differ as to (1) whether Bub1 recruits Mad1-Mad2 to kinetochores and (2) whether and how Bub1 contributes to SAC arrest [4-7, 9, 41]. Recently several groups reported that the SAC in human cells is refractory not only to Bub1 depletion [4,6] but also stable disruption of the BUB1 locus [9, 46] unless Mps1 is partially inhibited to sensitize the system [9, 47].…”

“…http://dx.doi.org/10.1101/297580 doi: bioRxiv preprint first posted online Apr. 9, 2018; 10 assembly downstream of Mad1-Mad2 [4]; however network-level compensation allows the SAC to adapt to chronic BUB1 loss, similar to other signaling pathways [48,49].…”

“…Recent studies have reached different conclusions about the specific roles and relative importance of Bub1 and the RZZ complex in targeting Mad1-Mad2 to kinetochores and transducing SAC arrest [4][5][6][7][8]. To interrogate RZZ function with maximum penetrance, we used AAV (adeno-associated virus)-and CRISPR-mediated gene editing to target the KNTC1 (Rod) locus in HCT116 cells, a diploid colorectal cell line ( Fig.…”

Bub1/BubR1: swiss army knives at kinetochores

“…The ABBA motif in Bub1 is required for a functional checkpoint and recruits a pool of Cdc20 to kinetochores. 3,4 Potentially the proximity of Cdc20 to the Mad1-Mad2 complex, coordinated by Bub1, facilitates the binding of Mad2 to Cdc20 the ratelimiting step in MCC formation.…”