Jcb_201407074 1..16

The kinetochore is a multilayered protein assembly on centromeric chromatin and acts as a platform on sister chromatids for the attachment of spindle microtubules during mitosis (Cleveland et al., 2003). The constitutive centromere-associated network (CCAN) of proteins binds to centromeric chromatin throughout the cell cycle and forms the inner kinetochore (Foltz et al., 2006; Okada et al., 2006; Black and Cleveland, 2011; Takeuchi and Fukagawa, 2012). It provides the foundation for the mitosisspecific assembly of the outer kinetochore. Among the outer kinetochore proteins, the KMN network, which consists of the Knl1 complex (Knl1C, which consists of Knl1 and Zwint in humans), the Mis12 complex (Mis12C, which comprises Dsn1, Nsl1, Mis12, and Nnf1), and the Ndc80 complex (Ndc80C, which comprises Ndc80, Nuf2, Spc25, and Spc24), acts as a receptor for spindle microtubules (Cheeseman et al., 2006; Cheeseman and Desai, 2008). In this network, Mis12C directly binds to both Knl1 and Ndc80C, thus bridging an interaction between the two (Gascoigne and Cheeseman, 2013; Petrovic et al., 2014). Accurate chromosome segregation relies on proper kinetochore–microtubule attachment during mitosis, which entails the capturing of a pair of sister kinetochores by microtubules originating from the two opposite spindle poles (a state termed bi-orientation; Cheeseman and Desai, 2008). Unattached or improperly attached kinetochores activate the spindle checkpoint to delay anaphase onset (Lara-Gonzalez et al., 2012; Foley and Kapoor, 2013; Jia et al., 2013). Furthermore, the centromeric kinase Aurora B severs improper kinetochore–microtubule attachments through phosphorylating multiple KMN components (Tanaka et al., 2002; Ruchaud et al., 2007; Welburn et al., 2010; Lampson and Cheeseman, 2011), thus promoting sister-chromatid bi-orientation. After all pairs of sister kinetochores reach bi-orientation, the spindle checkpoint is inactivated to allow synchronous dissolution of sister-chromatid cohesion and equal partition of the separated sister chromatids into the two daughter cells. In addition to microtubule binding, KMN recruits spindle checkpoint proteins to outer kinetochores during mitosis. Knl1 is the kinetochore receptor for the Bub1–Bub3 and BubR1– Bub3 checkpoint complexes (London et al., 2012; Shepperd et al., 2012; Yamagishi et al., 2012; Primorac et al., 2013; Vleugel et al., 2013; Krenn et al., 2014). Ndc80C is required for the During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sisterchromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMNinactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosisspecific assembly of the checkpoint platform KMN at human kinetochores. Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores