HSET motor function during mammalian mitosis and meiosis

Microtubule motors are essential for cell division. Although the functions of some mitotic motor proteins have been elucidated, the precise mechanisms by which they govern spindle assembly and chromosome segregation remain largely unknown. This thesis documents a program of research conducted to uncover the role of the human KIN C (Kinesin-related C-terminal motor) family member, HSET, in mammalian mitosis and meiosis. Prior studies describe various aspects of KIN C motor function in other species, and these motors are key players in mitotic and meiotic cell division. Therefore, it was hypothesized that HSET provides critical minus end-directed motor activity to mitotic and meiotic spindles in mammalian cells. This was tested using in vitro and in vivo techniques. It was discovered that two isoforms of HSET are generated from a single gene by alternative splicing which generates proteins with alternative C-termini. HSET-H (HSET-High) and HSET-L (HSET-Low) are differentially expressed in diverse cell types implying that each isoform has unique functional properties. Collective inhibition of both isoforms has deleterious effects on the formation and maintenance of asters assembled in cell free mitotic extracts. However specific inhibition of HSET-H has minor effects suggesting that HSET-L contributes the predominant HSET motor activity for aster assembly. Indeed, a fraction of HSET-L was found to be a component of a 15S complex that stably associates with microtubule asters. Inhibition of both isoforms also disrupts meiotic spindle formation but has no discernible effect on mitotic spindle organization demonstrating that centrosomes at mitotic spindle poles compensate for loss of HSET activity. During meiosis, HSET inhibition causes spindle poles to splay and fragment into discreet foci signifying that HSET principally focuses a subset of microtubule minus ends at poles. Together with the observation that HSET localizes between parallel microtubules in metaphase mitotic spindles, this implies that the primary role of HSET is to cross-link kinetochore fibers within mitotic and meiotic spindles. Therefore HSET has a novel role in mammalian cells, which stabilizes the framework of the spindle and contributes to formation of the spindle pole by stably connecting microtubule bundles and preventing their dissociation from one another.