Dimeric Centromere Protein E (CENP-E) Promotes Microtubule-elongation At The Plus-ends Of Microtubules

Abstract

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis including establishment of microtubule (MT)-chromosome linkage, processive movement of monooriented chromosomes on MTs for proper alignment at metaphase, and as a tension sensor at mitotic checkpoint to signal onset to anaphase. Fluorescence microscopy studies were performed to test the hypothesis that CENP-E promotes MT-elongation at the MT plus-ends. CENP-E constructs were engineered, expressed, and purified which yielded dimeric and monomeric motor proteins. The results show that dimeric CENP-E promotes plus-end directed MT gliding at 11 ± 0.005 nm/sec (n=173 MTs). Real-time microscopy assays were performed to image CENP-E promoted elongation of GMPcPP-stabilized polarity marked FITC MTs. The results revealed that out of the 270 polarity marked MTs examined, 164 MTs (60%) exhibited CENP-E promoted MT plus-end extension by GTP-tubulin (1.48 ± 0.37 μm/30 min; n=200 MTs) in the presence of MgATP. In contrast, dimeric Kinesin-1, dimeric Eg5, and CENP-E in the presence of AMPPNP did not show this pronounced MT elongation. These results suggest that CENP-E as part of its function for chromosome kinetochore attachment to MTs plays a direct role in kinetochore MT extension during congression. Supported by NIH GM54141 and NIH Career Development Award K02-AR47841 to SPG.