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CENP-F is a novel microtubule-binding protein that is essential for kinetochore attachments and affects the duration of the mitotic checkpoint delay

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Abstract

Centromeric protein F (CENP-F) is a 367-kDa human kinetochore protein that was identified a decade ago, but its function was only recently revealed by studies that used small interfering RNA to deplete the protein from cells. All studies showed that CENP-F is important for chromosome alignment, but these studies differed as to whether CENP-F is important to the mitotic checkpoint. We report here that CENP-F is essential for cells to sustain a prolonged mitotic delay in response to unattached kinetochores. Cells depleted of CENP-F exit mitosis in the presence of defective kinetochore attachments resulting from treatment with nocodazole, or the depletion of kinetochore proteins CENP-E and hSgo1. Kinetochores depleted of CENP-F exhibited a reduction in the amounts of the mitotic checkpoint proteins Mad1, Mad2, hBUBR1, hBUB1, and hMps1. We postulate that CENP-F is not an essential component of the mitotic checkpoint but facilitates the duration of the mitotic delay. Separately, we show that CENP-F is a novel microtubule-binding protein that possesses two microtubule-binding domains at opposite ends of the molecule. The C-terminal microtubule-binding domain was found to stimulate microtubule polymerization in vitro. These activities provide a biochemical explanation for how CENP-F contributes to kinetochore attachments in vivo.

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Acknowledgements

We acknowledge the excellent technical support by J. Hittle and B. Conner, and advice from S.T. Liu and other members of the Yen lab. We give special thanks to J. Peterson for the use of equipment. This work is supported by the National Institute of Health GM44762, core grants CA06927 CA75138, and an appropriation from the Commonwealth of Pennsylvania.

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Authors and Affiliations

  1. Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA, 19111, USA

    J. Feng, H. Huang & T. J. Yen

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  1. J. Feng
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  2. H. Huang
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  3. T. J. Yen
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Correspondence to T. J. Yen.

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Communicated by E.A. Nigg

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Fig8

CENP-F is not essential for checkpoint proteins to assemble onto kinetochores during prometaphase. Hela cells transfected with control and CENP-F siRNAs were fixed and stained for CENP-F and Bub1, BubR1, MPS1, Mad1 or Mad2. Scale bar: 10 μm. Cells in prometaphase were selected and only those whose kinetochores were depleted of CENP-F by >25-fold were chosen for analysis. The strongest signal detected at a control kinetochore was chosen as 100%. Approximately 150 kinetochores were quantified for each antibody. Error bars represent s.e.m

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Feng, J., Huang, H. & Yen, T.J. CENP-F is a novel microtubule-binding protein that is essential for kinetochore attachments and affects the duration of the mitotic checkpoint delay. Chromosoma 115, 320–329 (2006). https://doi.org/10.1007/s00412-006-0049-5

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