KNL1-Bubs and RZZ Provide Two Separable Pathways for Checkpoint Activation at Human Kinetochores

Abstract

The spindle assembly checkpoint (SAC) ensures the accurate segregation of sister chromatids during mitosis. Activation of the SAC occurs through a series of ordered molecular events that result in recruitment of Mad1:Mad2 complexes to improperly attached kinetochores. The current model involves sequential phospho-dependent recruitment of Bub3:Bub1 to KNL1 followed by binding of Mad1:Mad2 to Bub1. Here, we show in non-transformed diploid human cells that the KNL1-Bub3-Bub1 (KBB) pathway is required during normal mitotic progression when kinetochores are misaligned but is nonessential for SAC activation and Mad2 loading when kinetochores are unattached from microtubules. We provide evidence that the Rod-ZW10-Zwilch (RZZ) complex is necessary to recruit Mad1:Mad2 to, and delay anaphase onset in response to, unattached kinetochores independently of the KBB pathway. These data suggest that the KBB and RZZ complexes provide two distinct kinetochore receptors for Mad1:Mad2 and reveal mechanistic differences between SAC activation by unattached and improperly attached kinetochores.

Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects

“…Although cell-cycle processes are required for the duplication of all cells, we next considered the possibility that the precise requirements for faithful cell division may differ between cell types (for example, see Salimian et al, 2011; Silio et al, 2015). Therefore, we sought to determine whether the phenotypes that we defined for cell-cycle-associated genes in HeLa cells vary across cellular backgrounds.…”

Heterogeneous architecture of vertebrate kinetochores revealed by three-dimensional superresolution fluorescence microscopy

“…During mitosis, the CCAN further recruits a network composed of the K NL1 complex (KNL1C), the M is12 complex (Mis12C), and the Ndc80 complex (Ndc80C; the KMN complex). Ndc80C is responsible for microtubule end-on attachment, whereas the KNL1 protein acts as a major scaffold for the transient spindle assembly checkpoint (SAC) proteins (including Bub1, BubR1, Bub3, Mad1, Mad2, the Rod/ZW10/Zwilch [RZZ] complex, and Spindly; Varma and Salmon, 2012; Silio et al ., 2015) and the motor proteins dynein and CENP-E, which capture the lateral sides of microtubules (Rieder and Alexander, 1990; Wood et al ., 1997; Kapoor et al ., 2006). These motors and checkpoint proteins are localized to a “fibrous corona” region that can extend >100 nm into the cytoplasm from the outer kinetochore plate (McEwen et al ., 1998; Hoffman et al ., 2001).…”

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

“…http://dx.doi.org/10.1101/297580 doi: bioRxiv preprint first posted online Apr. 9, 2018; microtubule attachment [7]. However KNTC1-null RPE cells exhibited severe SAC failure when kinetochore attachments were ablated with nocodazole or when STLC or taxol were used to induce erroneous attachments (Fig.…”

“…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.…”

“…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.…”