Mitotic Kinesin CENP-E Promotes Microtubule Plus-End Elongation

Abstract

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 monooriented chromosomes on kinetochore microtubules (kMTs) 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 which 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 μm/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 (kobs = 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.

Interhead tension determines processivity across diverse N-terminal kinesins

“…This run length is within 15% of the value measured by Kim et al (27) in a comparable ionic strength buffer and it is half of that measured by Yardimci et al (28) at low ionic strength (10 mM Pipes). We also made a similar construct using the head and neck linker of human CENP-E and found the motor moved at 10 nm∕s, consistent with results from Gilbert lab (32). This result suggests that the slow speeds of human CENP-E are due to slow ATP hydrolysis or some other step in the hydrolysis cycle rather than misregistration of the coiled coil as has been suggested (5).…”

“…In single-molecule fluorescence experiments, Xenopus CENP-E has been shown to be as processive as kinesin-1 and to generate comparable stall forces (27,28). Disagreements in measured CENP-E motor speed and processivity have been explained by suggesting that the CENP-E coiled-coil region may possess a weak propensity for dimerization, may interact electrostatically with the microtubule to enhance processivity, or may sterically inhibit the motor domains in a phosphorylation-dependent manner (26)(27)(28)32). Hence, fusing the Xenopus CENP-E head and neck linker domain to the stable and well-characterized kinesin-1 coiled coil is an ideal approach for clearly defining the motile properties of CENP-E.…”

Prime movers: the mechanochemistry of mitotic kinesins

“…Indeed, full length and truncated KIF10 dimers can track growing microtubule tips for several seconds 128,129 and full length KIF10 can track shrinking tips, requiring both motor activity and a C-terminal nonmotor microtubule binding site to do so 128 . There are also data suggesting that KIF10 may influence the dynamics of kinetochore-attached microtubules: a truncated human KIF10 construct accelerates microtubule growth in the presence of low concentrations of taxol 130 .…”

CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment

“…To distinguish between the role of CENP-E-mediated forces on congressing chromosomes and other possible motor-dependent activities of CENP-E (for example, promotion of microtubule plus-end elongation associated with spindle microtubule flux 26 ) as the cause of spindle multipolarity in CLASP1/2-depleted cells, we used a reversible allosteric inhibitor of CENP-E, GSK-923295, which allows temporal control of CENP-E motor activity 27 . First, we used GSK-923295 in a series of inhibition-washout experiments in living cells stably co-expressing H2B-histone-GFP and α-tubulin-mRFP.…”