Efficient mitotic checkpoint signaling depends on integrated activities of Bub1 and the RZZ complex

Abstract

Kinetochore localized Mad1 is essential for generating a “wait anaphase” signal during mitosis, hereby ensuring accurate chromosome segregation. Inconsistent models for the function and quantitative contribution of the two mammalian Mad1 kinetochore receptors: Bub1 and the Rod‐Zw10‐Zwilch (RZZ) complex exist. By combining genome editing and RNAi, we achieve penetrant removal of Bub1 and Rod in human cells, which reveals that efficient checkpoint signaling depends on the integrated activities of these proteins. Rod removal reduces the proximity of Bub1 and Mad1, and we can bypass the requirement for Rod by tethering Mad1 to kinetochores or increasing the strength of the Bub1‐Mad1 interaction. We find that Bub1 has checkpoint functions independent of Mad1 localization that are supported by low levels of Bub1 suggesting a catalytic function. In conclusion, our results support an integrated model for the Mad1 receptors in which the primary role of RZZ is to localize Mad1 at kinetochores to generate the Mad1‐Bub1 complex.

Nek2 kinase displaces distal appendages from the mother centriole prior to mitosis

“…7, A and B;Ishikawa et al, 2005;Tateishi et al, 2013;Mazo et al, 2016). Furthermore, RT-PCR of RPE1 Nek2 KO cells indicated that all sequenced transcripts contained the mutated exon (data not shown), excluding the possibility of exon skipping (Uddin et al, 2015;Rodriguez-Rodriguez et al, 2018;Meraldi, 2019;Zhang et al, 2019). Interestingly, the levels of DAs at interphase centrosomes remained unchanged in ODF2 KO cells ( Fig.…”

Untangling the contribution of Haspin and Bub1 to Aurora B function during mitosis

“…Of note, we chose to inhibit Bub1 kinase activity instead of depleting Bub1, because Bub1 also has nonenzymatic functions, including the recruitment of mitotic checkpoint proteins to the kinetochore, a function that is maintained after inhibition of its kinase activity (Baron et al, 2016; Meraldi and Sorger, 2005; Raaijmakers et al, 2018; Rodriguez-Rodriguez et al, 2018; Zhang et al, 2019). As previously reported, treatment of cells with the Bub1 inhibitor BAY-320 markedly reduced H2AT120ph and Sgo1 levels at the centromere (Fig.…”

Killing a zombie: a full deletion of the BUB 1 gene in HAP 1 cells

“…While some studies confirmed a (near)-essential role for BUB1 in the SAC (Meraldi & Sorger, 2005;Klebig et al, 2009), other studies reported no obvious SAC defect in Bub1deficient cells (Johnson, 2004) or showed a SAC defect that could only be observed after sensitization with inhibitors for Mps1 (Vleugel et al, 2015). Recently, Zhang et al combined CRISPR/ Cas9-mediated gene editing with siRNA depletion and concluded that BUB1 also makes a prominent contribution to the SAC in HeLa cells (Zhang et al, 2019). We and others showed that deletion of BUB1 in HAP1 or RPE-1 cells resulted in only a minor SAC defect (Currie et al, 2018;Raaijmakers et al, 2018), while another study showed that (acute) deletion of BUB1 in p53-deficient RPE-1 cells results in a more prominent SAC defect (Rodriguez-Rodriguez et al, 2018).…”