By using monoclonal antibodies raised against isolated clam centrosomes, we have identified a novel 135-kD centrosomal protein (Cep135), present in a wide range of organisms. Cep135 is located at the centrosome throughout the cell cycle, and localization is independent of the microtubule network. It distributes throughout the centrosomal area in association with the electron-dense material surrounding centrioles. Sequence analysis of cDNA isolated from CHO cells predicted a protein of 1,145–amino acid residues with extensive α-helical domains. Expression of a series of deletion constructs revealed the presence of three independent centrosome-targeting domains. Overexpression of Cep135 resulted in the accumulation of unique whorl-like particles in both the centrosome and the cytoplasm. Although their size, shape, and number varied according to the level of protein expression, these whorls were composed of parallel dense lines arranged in a 6-nm space. Altered levels of Cep135 by protein overexpression and/or suppression of endogenous Cep135 by RNA interference caused disorganization of interphase and mitotic spindle microtubules. Thus, Cep135 may play an important role in the centrosomal function of organizing microtubules in mammalian cells.
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7 January 2002
Article|
January 07 2002
Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells
Toshiro Ohta,
Toshiro Ohta
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
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Russell Essner,
Russell Essner
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
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Jung-Hwa Ryu,
Jung-Hwa Ryu
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
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Robert E. Palazzo,
Robert E. Palazzo
2Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045
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Yumi Uetake,
Yumi Uetake
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
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Ryoko Kuriyama
Ryoko Kuriyama
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
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Toshiro Ohta
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
Russell Essner
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
Jung-Hwa Ryu
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
Robert E. Palazzo
2Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045
Yumi Uetake
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
Ryoko Kuriyama
1Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455
Address correspondence to Ryoko Kuriyama, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. S.E., University of Minnesota, Minneapolis, MN 55455. Tel.: (612) 624-0471. Fax: (612) 626-6140. E-mail: [email protected]
*
Abbreviations used in this paper: GFP, green fluorescence protein; HA, hemagglutinin; MAP, microtubule-associated proteins; MTOC, microtubule organizing center; pBS, pBluescript; PBS-Tw20, Tween-20–containing PBS; siRNA, small interfering RNA; RNAi, RNA interference.
Received:
August 17 2001
Revision Received:
November 08 2001
Accepted:
November 21 2001
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2002
J Cell Biol (2002) 156 (1): 87–100.
Article history
Received:
August 17 2001
Revision Received:
November 08 2001
Accepted:
November 21 2001
Citation
Toshiro Ohta, Russell Essner, Jung-Hwa Ryu, Robert E. Palazzo, Yumi Uetake, Ryoko Kuriyama; Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells . J Cell Biol 7 January 2002; 156 (1): 87–100. doi: https://doi.org/10.1083/jcb.200108088
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