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First published online 26 June 2003
doi: 10.1242/jcs.00645
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Research Article |
1 PRESTO, The Japan Science and Technology Corporation, National Institute of
Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka
411-8540, Japan
2 CREST Research Project, The Japan Science and Technology Corporation, Kansai
Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka,
Nishi-ku, Kobe 651-2492, Japan
3 Department of Functional Genomics, Medical Research Institute, Tokyo Medical
and Dental University, Tokyo 113-8510, Japan
* Author for correspondence (e-mail: tfukagaw{at}lab.nig.ac.jp)
Accepted 1 May 2003
Nuf2 and Hec1 are evolutionarily conserved centromere proteins. To clarify the functions of these proteins in vertebrate cells, we characterized them in chicken DT40 cells. We generated GFP fusion constructs of Nuf2 and Hec1 to examine in detail the localization of these proteins during the cell cycle. We found that Nuf2 is associated with Hec1 throughout the cell cycle and that this complex is localized to the centrosomes during G1 and S phases and then moves through the nuclear membrane to the centromere in G2 phase. During mitosis, this complex is localized to the centromere.
We also created conditional loss-of-function mutants of Nuf2 and Hec1. In both mutants, the cell cycle arrested at prometaphase, suggesting that the Nuf2-Hec1 complex is essential for mitotic progression. The inner centromere proteins CENP-A, -C, and -H and checkpoint protein BubR1 were localized to chromosomes in the mutant cells arrested at prometaphase, but Mad2 localization was abolished. Furthermore, photobleaching experiments revealed that the Nuf2-Hec1 complex is stably associated with the centromere and that interaction of this complex with the centrosome is dynamic.
Key words: Nuf2, Hec1, Centrosome, Centromere, FRAP
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