Analysis of the Kinesin-13 gene family in plants

Abstract

Although the depolymerizing effects of animal kinesin-13 proteins have been well characterized, little is known of the two Arabidopsis kinesin-13 proteins. This project aimed to investigate in more detail the functions of AtKinesin-13A/B and their effect on microtubule dynamics. The first aim was to analyse the function of both proteins in vitro using biochemical studies. Proteins were expressed in both bacterial and insect cells before undergoing co-sedimentation and turbidimetric assays to assess their activity and microtubule-interacting properties. The second aim of this project was to investigate the in vivo effects of these proteins on microtubule dynamics throughout the cell cycle. Tobacco BY2 cell lines were transformed with AtKinesin-13A and 13B, as well as human HsKinesin-13 for comparison under the expression of an estradiol-inducible promoter. This inducible system allowed transient expression of recombinant protein to analyse the effect of expression on cell growth, nuclear morphology and microtubule dynamics by immunofluorescence microscopy.

These experiments show that AtKin13B appears to function during metaphase in aiding activation of the metaphase spindle checkpoint and subsequent progression through the cell cycle. Biochemical studies into its relationship with microtubules, combined with in vivo induction data, suggest that AtKin13B may have a subtle destabilizing effect on microtubules. Biochemical studies with AtKin13A showed that it binds specifically to microtubules, and it localized to chromosome arms in a similar pattern to AtKin13B, but may have different effects in plants. Human Kinesin-13 localized exclusively to spindle microtubules throughout the cell cycle showing that the activity of kinesin-13 proteins is species-specific and animal kinesins have little effect in plant systems.