期刊
CURRENT BIOLOGY
卷 20, 期 18, 页码 1648-1653出版社
CELL PRESS
DOI: 10.1016/j.cub.2010.08.001
关键词
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资金
- GenNYsis
- National Institutes of Health [GM54141]
Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis, including establishment of microtubule (MT)-chromosome linkage and movement of mono-oriented chromosomes on kinetochore microtubules for proper alignment at metaphase [1-9]. We performed studies to test the hypothesis that CENP-E promotes MT elongation at the MT plus ends. A human CENP-E construct was engineered, expressed, and purified, and it yielded the CENP-E-6His dimeric motor protein. The results show that CENP-E promotes MT plus-end-directed MT gliding at 11 nm/s. The results from real-time microscopy assays indicate that 60.3% of polarity-marked MTs exhibited CENP-E-promoted MT plus-end elongation. The MT extension required ATP turnover, and MT plus-end elongation occurred at 1.48 mu m/30 min. Immunolocalization studies revealed that 80.8% of plus-end-elongated MTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongation in solution best fit a single exponential function (k(obs) = 5.1 s(-1)), which is indicative of a mechanism in which alpha, beta-tubulin subunit addition is tightly coupled to ATP turnover. Based on these results, we propose that CENP-E, as part of its function in chromosome kinetochore-MT linkage, plays a direct role in MT elongation.
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