CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

doi:10.1083/jcb.139.6.1373

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© The Rockefeller University Press, 0021-9525/1997//1373 $5.00
The Journal of Cell Biology, Volume 139, Number 6, , 1997 1373-1382

Article

CENP-E Function at Kinetochores Is Essential for Chromosome Alignment

B.T. Schaar^*, G.K.T. Chan, P. Maddox, E.D. Salmon, and T.J. Yen

^* Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19103; Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599; and Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

CENP-E is a kinesin-like protein that binds to kinetochoresand may provide functions that are critical for normal chromosomemotility during mitosis. To directly test the in vivo functionof CENP-E, we microinjected affinity-purified antibodies toblock the assembly of CENP-E onto kinetochores and then examinedthe behavior of these chromosomes. Chromosomes lacking CENP-Eat their kinetochores consistently exhibited two types of defectsthat blocked their alignment at the spindle equator. Chromosomes positioned near a pole remained mono-oriented as they wereunable to establish bipolar microtubule connections with theopposite pole. Chromosomes within the spindle established bipolarconnections that supported oscillations and normal velocitiesof kinetochore movement between the poles, but these bipolarconnections were defective because they failed to align thechromosomes into a metaphase plate.

Overexpression of a mutant that lacked the amino-terminal 803amino acids of CENP-E was found to saturate limiting bindingsites on kinetochores and competitively blocked endogenousCENP-E from assembling onto kinetochores. Chromosomes saturated with the truncated CENP-E mutant were never found to be alignedbut accumulated at the poles or were strewn within the spindleas was the case when cells were microinjected with CENP-E antibodies.As the motor domain was contained within the portion of CENP-Ethat was deleted, the chromosomal defect is likely attributedto the loss of motor function.

The combined data show that CENP-E provides kinetochore functionsthat are essential for monopolar chromosomes to establish bipolarconnections and for chromosomes with connections to both spindlepoles to align at the spindle equator. Both of these eventsrely on activities that are provided by CENP-E's motor domain.

Abbreviations used in this paper: ACA, anti-centromere autoimmune serum; DIC, differential interference contrast; GFP, green fluorescent protein; GST, glutathione-S-transferase; MBP, maltose-binding protein; VE, video enhanced.

Address all correspondence to Tim J. Yen, Fox Chase Cancer Center,770 Burholme Avenue, Philadelphia, PA 19111. Tel.: (215) 728-2590.Fax: (215) 728-3616.

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