Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

Abstract

Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the γ-tubulin ring complex (γ-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to γ-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at γ-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.

Molecular properties of the N-terminal extension of the fission yeast kinesin-5, Cut7

“…A recent report revealed that Cut7 regulates microtubule nucleation at spindle poles, and that its head and tail bind to the g-TuRC (Olmsted et al, 2014). Considering the localization of Cut7-DN2, the N-terminal extension is unlikely to be involved in such g-TuRC binding.…”

“…Considering the localization of Cut7-DN2, the N-terminal extension is unlikely to be involved in such g-TuRC binding. The mechanism by which microtubule nucleation at spindle poles is regulated is thought to be conserved in human cells (Olmsted et al, 2014), and thus Eg5 (human kinesin-5) might associate with the g-TuRC in a similar manner to Cut7.…”

“…However, a recent report revealed that Cut7 moves toward both the minus and plus ends of microtubules (Edamatsu, 2014), as with budding yeast kinesin-5 (Roostalu et al, 2011;Fridman et al, 2013). Furthermore, it was recently shown that Cut7 regulates the microtubule-nucleating activity of the g-tubulin ring complex (g-TuRC) at spindle poles through an antagonistic relationship with pkl1 (fission yeast kinesin-14; Olmsted et al, 2014). As the properties of the Cut7 motor are of current interest, this study aimed to investigate the molecular characteristics of the N-terminal extension of this protein using in vivo and in vitro analyses.…”

Molecular mechanisms of kinesin-14 motors in spindle assembly and chromosome segregation

“…KIFC5A has an additional ATP-independent microtubule-binding domain (Zhang and Sperry, 2004) and Ncd has a positively charged sequence in its tail domain that tethers it to the C-terminal E-hook of tubulin . Mouse KIFC1 has tail domain sequences that target it to membrane-bound organelles (Zhang and Sperry, 2004); the tail domain of Pkl1 direct it to γ-tubulin (Olmsted et al, 2013(Olmsted et al, , 2014. Additional studies in which chimeric kinesin-14 proteins of human HSET, Drosophila Ncd and fission yeast Pkl1 were generated also support this paradigm and confirm that tail domain sequences orchestrate function (Simeonov et al, 2009).…”

“…An exception to this is Pkl1, for which the tail domain was shown to be able to regulate nucleation without the motor domain in vitro and in vivo (Fig. 1B), and to do so in yeast and human cells (Olmsted et al, 2013(Olmsted et al, , 2014. Microtubule-dependent D. melanogaster Ncd motors have been shown to localize to parallel microtubules where they exert their movement into the opposite direction, thus creating a 'tug-of-war' effect.…”