Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Summary 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 kinetochoremicrotubules for proper alignment at metaphase [1–9]. We performed studies to test the hypothesis that CENP-E promotes MT elongationat 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 elongationoccurred at 1.48 μm/30 min. Immunolocalization studies revealed that 80.8% of plus-end- elongatedMTs showed CENP-E at the MT plus end. The time dependence of CENP-E-promoted MT elongationin solution best fit a single exponential function ( k obs = 5.1 s −1 ), which is indicative of a mechanism in which α,β-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.