BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised

Abstract

The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment.

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion

“…How (and why) this sustained response occurs is not well understood. In yeast, Bub1 is the sole receptor for Mad1-Mad2 and required for the SAC [37], but models for Mad1-Mad2 regulation in mammalian cells differ considerably [7–12]. In our studies, acute BUB1 or KNL1 deletion led to SAC failure despite high levels of Mad1-Mad2 at kinetochores.…”

“…Bub1’s role in the SAC also remains controversial, with inconsistent results across studies [7–12]. To test Bub1’s contribution to these aspects of kinetochore structure and function, we deleted BUB1 in RPE cells via doxycycline-inducible CRISPR/Cas9 [27].…”

“…To test Bub1’s contribution to these aspects of kinetochore structure and function, we deleted BUB1 in RPE cells via doxycycline-inducible CRISPR/Cas9 [27]. BUB1 –/– cells treated with nocodazole formed kinetochore crescents containing Rod, CENP-E, and Mad1, but not CENP-F [12] (Figure 3A and Figure S3A). To ensure complete depletion and avoid postmitotic arrest [27], we deleted BUB1 or its kinetochore scaffold KNL1 [1, 2] in p53-deficient RPE cells.…”

“…Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4–6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7–12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells.…”

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

“…We refer to this biochemical cascade as the ‘core SAC signaling cascade’ (dashed gray box in Figure 1B). In metazoa, the core SAC signaling cascade is complemented by the RZZ pathway, which independently recruits additional Mad1-Mad2 to the kinetochore [20]. Numerous additional kinases and phosphatases provide additional regulation to these interdependent interactions, but they are not shown here.…”