Mechanistic and functional insights into the human kinesin motor CENP-E in cell division

Abstract

During mitosis, chromosomes align at the spindle equator and biorient in order to equally distribute the genome into two daughter cells. A macromolecular protein complex, known as the kinetochore, facilitates the end-on attachment of chromosomes to spindle microtubules. CENP-E is a very large mitotic kinesin motor protein which is recruited to the outer kinetochore and fibrous corona of unattached kinetochores in prometaphase. Human CENP-E motor activity is essential for the alignment of chromosomes close to the spindle poles, but also for the stabilisation of kinetochore-microtubule attachments and microtubule flux in the mitotic spindle. Until now, biochemical characterisation studies and reconstitutions of CENP-E activity have used the Xenopus laevis CENP-E orthologue as a model motor. However, human and X. laevis CENP-E share only 49% sequence similarity and the human model system is typically used for cell biology, functional and structural studies of human kinetochores. The aim of my thesis was to define the mechanistic properties of human CENP-E and define how interactions with associated proteins direct its function in mitosis. First, I reconstituted motor activity of truncated and full-length human CENP-E using reconstitution approaches and single molecule imaging. Truncated CENP-E is constitutively active and processive in vitro, capable of unidirectional movement along microtubules. Active full-length CENP-E molecules are more processive than their truncated CENP-E counterparts in vitro, but exhibit slower average speeds and lower landing rates on microtubules. This work indicates that the non-motor regions of human CENP-E contribute to the regulation of motor activity. CENP-E has been suggested to interact with several distinct binding partners, but it is unclear whether many of these reported interactions are direct. Using biochemistry and isothermal titration calorimetry (ITC), I reconstituted binding between human CENP-E and Protein Phosphatase 1 (PP1). Finally, I studied the role of CENP-E at the spindle midzone. As cells progress into anaphase and the chromosomes segregate to opposite poles, CENP-E is gradually lost from kinetochores and relocalises to the midzone in a PRC1-dependent manner. Thus, I used in vitro reconstitution approaches to gain molecular insights into the function of CENP-E at the overlapping microtubule bundles of the spindle midzone and midbody. I demonstrated that PRC1 is able to recruit CENP-E to overlapping microtubule bundles. PRC1 facilitates microtubule sliding activity of CENP-E in vitro, providing important molecular insight into how CENP-E contributes to microtubule flux and organisation of the spindle midzone in vivo. This study defines the molecular properties of human CENP-E which underpin the essential functions of the motor in chromosome transport, kinetochore-microtubule attachments and mitotic spindle organisation in vivo.