Reconstitution of an active human CENP-E motor

Abstract

CENP-E is a large kinesin motor protein which plays pivotal roles in mitosis by facilitating chromosome capture and alignment, and promoting microtubule flux in the spindle. So far, it has not been possible to obtain active human CENP-E to study its molecular properties. Xenopus CENP-E motor has been characterized in vitro and is used as a model motor; however, its protein sequence differs significantly from human CENP-E. Here, we characterize human CENP-E motility in vitro . Full-length CENP-E exhibits an increase in run length and longer residency times on microtubules when compared to CENP-E motor truncations, indicating that the C-terminal microtubule-binding site enhances the processivity when the full-length motor is active. In contrast with constitutively active human CENP-E truncations, full-length human CENP-E has a reduced microtubule landing rate in vitro , suggesting that the non-motor coiled-coil regions self-regulate motor activity. Together, we demonstrate that human CENP-E is a processive motor, providing a useful tool to study the mechanistic basis for how human CENP-E drives chromosome congression and spindle organization during human cell division.

Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis

“…We previously reconstituted motility of both truncated and full-length CENP-E in vitro (Craske et al, 2022). The challenge in analyzing the contribution of CENP-E to microtubule sliding is that about 10% purified full-length CENP-E is motile, with the long coiled-coil stalk interfering with its activity (Fig S2A)(Craske et al, 2022). In order to determine whether CENP-E slides microtubules alone, or only slides those crosslinked by PRC1, we first analyzed whether CENP-E was recruited to microtubules crosslinked with PRC1, or to PRC1 directly.…”

“…Together, these data suggest that a CENP-E-PRC1 complex is able to slide antiparallel microtubules relative to each other. The sliding velocity may be regulated by frictional forces that are either generated by the accumulation over time of PRC1 on cross-linked microtubules, similar to Kif4A/PRC1 sliding (Wijeratne and Subramanian, 2018), or by the fraction of inactive or paused microtubule-bound CENP-E motors that can still bind to PRC1 (Craske et al, 2022).…”

“…The timing of the PRC1:motor interaction is important, because Kif4A and CENP-E are involved in chromosome organization and alignment in early mitosis (reviewed in (Craske et al, 2022; Samejima et al, 2012). PRC1 is phosphorylated by CDK1/cyclin B on T470 and T481, in the region important for microtubule binding, at the junction between the unstructured microtubule binding tail and the spectrin domain (Jiang et al, 1998).…”

“…Kinetochore-bound CENP-E enables kinetochore capture and lateral attachment to microtubules. CENP-E moves chromosomes along the spindle to the metaphase plate before kinetochore biorientation (Fig 1A), reviewed in (Craske et al, 2022). The CENP-E motor relocalizes from kinetochores to the central spindle, at the metaphase to anaphase transition (Kurasawa et al, 2004; Yao et al, 1997).…”

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona

“…One is the homo-dimeric plus-end-directed kinesin-7 centromere protein E (CENP-E, 2701 residues in humans) (Yen et al, 1991;Yen et al, 1992). Like other kinesins, CENP-E is autoinhibited, likely through an interaction of its N-and C-terminal regions that is regulated by Aurora kinase phosphorylation (Craske et al, 2022;Espeut et al, 2008;Kim et al, 2008;Kim et al, 2010;Vitre et al, 2014). How CENP-E autoinhibition regulates end-on attachment, however, remains unclear.…”