fected by immunodepletion procedures, this dramatic expansion provides an excellent system to characterize microtubule-dependent changes in kinetochore structure. The expandable module is composed of kinetochore components that promote lateral attachment and the SAC We next systematically examined which kinetochore submodules show expansion in the absence of microtubule attachment. Outermost kinetochore components, such as CENP-E, Xorbit, and dynein, were present throughout the expanded kinetochores. In addition, Mad1 and Mad2 were both stained heavily on the expanded kinetochore, despite being weakly present or undetectable on untreated kinetochores. In contrast, Ndc80 showed a slight increase in volume and intensity in nocodazole-treated Xenopus egg extracts, but it remained mostly at the core of each kinetochore and failed to robustly localize along the extended fibers. However, although Ndc80 is a member of the KMN network, other KMN components did expand: Mis12 and Zwint, which binds tightly to the C terminus of KNL1, were both found throughout the expanded region, and thus, substantial amounts of each protein are spatially segregated from the Ndc80C. CENP-C is a Oleandrin biological activity component of the expandable kinetochore Results Outer kinetochore proteins undergo dramatic expansion in the absence of microtubules To investigate the assembly of vertebrate kinetochores and their response to microtubule attachment status, we monitored kinetochore architecture using 3D SIM in Xenopus egg extracts, where kinetochore formation occurs on replicated sperm chromosomes in a synchronous manner. Although BubR1 forms 900 JCB Volume 210 NumBer 6 2015 The different behaviors among the KMN network raised the question of how the internal CCAN components would respond to the lack of microtubules. Consistent with its role in directly recruiting the Ndc80C, CENP-T remained in small foci in nocodazole-treated extracts. CENP-K, a component of the CENP-H/I/K complex contributing to Ndc80C recruitment, also remained in small foci. Surprisingly, CENP-C is present at high levels throughout the expanded kinetochore and, like BubR1, increases in both intensity and volume. We also observed an increase in CENP-C recovered on chromosomes purified from nocodazole-treated extracts. CENP-C harbors multiple well-characterized functional domains of which the central CENP-A interaction domain, CENP-C motif, and dimerization domain, but not the Mis12C-interacting domain, are required for CENP-C to localize to the kinetochore. To define the domains that target CENP-C to the expanded kinetochore, we expressed full-length and truncated versions of CENP-C containing six N-terminal Myc tags 
Kinetochore expansion and checkpoint activation Wynne and Funabiki 901 902 JCB Volume 210 NumBer 6 2015 in egg extracts. Although FL MycCENP-C was able to recapitulate the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834025 localization of endogenous CENP-C, a truncation mutant lacking either 380 amino acids or 711 amino acids of the N terminus was found almost exclusively at the CENP-Amarked core and not on the expanded region. In contrast, Myc-tagged N-terminal fragments lacking the C-terminal 109 amino acids responsible for dimerization were targeted to the expanded region containing BubR1. Shorter N-terminal fragments lacking the CENP-A interaction domain were also targeted to the expanded region but with much reduced efficiency. These results indicate that the N-terminal 380 amino acids of CENP-C, which interacts with the Mis12C, drives preferential tar
