Abstract
The spindle assembly checkpoint (SAC) arrests cells in mitosis by sensing unattached kinetochores, until all chromosomes are bi-oriented by spindle microtubules. Kinetochore accumulation of the SAC component Mad1–Mad2 is crucial for SAC activation. However, the mechanism by which Mad1–Mad2 accumulation at kinetochores is regulated is not clear. Here we find that Cep57 is localized to kinetochores in human cells, and binds to Mis12, a KMN (KNL1/Mis12 complex/Ndc80 complex) network component. Cep57 also interacts with Mad1, and depletion of Cep57 results in decreased kinetochore localization of Mad1–Mad2, reduced SAC signalling and increased chromosome segregation errors. We also show that the microtubule-binding activity of Cep57 is involved in the timely removal of Mad1 from kinetochores. Thus, these findings reveal that the KMN network-binding protein Cep57 is a mitotic kinetochore component, and demonstrate the functional connection between the KMN network and the SAC.
Highlights
The spindle assembly checkpoint (SAC) arrests cells in mitosis by sensing unattached kinetochores, until all chromosomes are bi-oriented by spindle microtubules
To determine whether Cep[57] is located at kinetochores in human cells, we raised a mouse polyclonal antibody against the Cep[57] protein (B60 kDa; Supplementary Fig. 1a)[34], which did not cross-react with Cep57-related protein (Cep57R) (Supplementary Fig. 1b)
To investigate the kinetochore localization of Cep[57] in detail, we further co-immunostained for Cep[57] and the inner kinetochore marker CENP-A39 or the KMN network component Mis[12]
Summary
The spindle assembly checkpoint (SAC) arrests cells in mitosis by sensing unattached kinetochores, until all chromosomes are bi-oriented by spindle microtubules. We show that the microtubule-binding activity of Cep[57] is involved in the timely removal of Mad[1] from kinetochores. These findings reveal that the KMN network-binding protein Cep[57] is a mitotic kinetochore component, and demonstrate the functional connection between the KMN network and the SAC. Cep[57] is required for centriole duplication, and is highly expressed in prostate cancer cells[37] Both xCep[57] and Cep[57] are localized at centrosomes[31,33,34,36], but whether Cep[57] is localized to kinetochores remains unclear. We propose that Cep[57] regulates the Mad1–Mad2-mediated monitoring of kinetochore-microtubule attachment
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