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Vol. 18, Issue 6, 2155-2168, June 2007
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Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies, Shizuoka 411-8540, Japan
Submitted January 22, 2007;
Revised March 8, 2007;
Accepted March 20, 2007
Monitoring Editor: Wendy Bickmore
CENP-C is a conserved inner kinetochore component. To understand the precise roles of CENP-C in the kinetochore, we created a cell line with a conditional knockout of CENP-C with the tetracycline-inducible system in which the target protein is inactivated at the level of transcription. We found that CENP-C inactivation causes mitotic delay. However, observations of living cells showed that CENP-C-knockout cells progressed to the next cell cycle without normal cell division after mitotic delay. Interphase cells with two nuclei before subsequent cell death were sometimes observed. We also found that 
60% of CENP-C–deficient cells had no Mad2 signals even after treatment with nocodazole, suggesting that lack of CENP-C impairs the Mad2 spindle checkpoint pathway. We also observed significant reductions in the signal intensities of Mis12 complex proteins at centromeres in CENP-C–deficient cells. CENP-C signals were also weak in interphase nuclei but not in mitotic chromosomes of cells with a knockout of CENP-K, a member of CENP-H complex proteins. These results suggest that centromere localization of CENP-C in interphase nuclei occurs upstream of localization of the Mis12 complex and downstream of localization of the CENP-H complex.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Tatsuo Fukagawa (tfukagaw{at}lab.nig.ac.jp).
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